Gip peptide analogues

ABSTRACT

Provided herein are glucose-dependent insulinotropic polypeptide (GIP)-derived peptide analogues, for example GIP(3-30), and their use as antagonists of the GIP receptor and for treatment of disorders such as obesity, diabetes mellitus and insulin resistance.

FIELD OF INVENTION

The present invention relates to Glucose-dependent insulinotropicpeptide-derived peptide analogues and their use for treatment ofdisorders such as obesity, diabetes mellitus and insulin resistance.

BACKGROUND OF INVENTION

Glucose-dependent insulinotropic peptide (GIP) is a hormone secretedfrom the K cells of the gut following a meal¹. Like its sister hormoneglucagon-like peptide 1 (GLP-1), GIP is a potent insulin secretagogue².In contrast to the glucagonostatic effect of GLP-1^(3,4), GIP has beenshown to display glucagon-releasing properties under certainconditions⁽³⁻⁵⁻¹³⁾ The interest in understanding the biology of GIP wasintensified by the association between rodent GIPR (GIP receptor) andadiposity^(14-17, 17-21). In humans, although less clear, there islikewise evidence for a role of GIP in fat metabolism with thedemonstration of the GIPR expression in adipose tissue²², an associationbetween high BMI and increased GIP levels^(22, 23), increased adiposetissue blood flow and TAG (triacylglycerol) deposition following GIPadministration in a state of high insulin and high glucose²⁴, decreasedbasal and postprandial GIP levels observed in obese children put on adiet²⁵, and increased fasting GIP levels observed in healthy young menput on a high fat diet²⁶.

Thus, in addition to the general demand from researchers who witnessedthe advances in the understanding of GLP-1 following the discovery ofthe GLP-1 receptor antagonist, exendin(9-39)^(27,28) the potential as ananti-obesity agent has attracted additional attention for thedevelopment of potent GIPR antagonists. Many different strategies havebeen undertaken in order to antagonize GIP's function, e.g. a smallmolecule receptor antagonist²⁹, immunization against GIP³⁰⁻³² varioustruncations and mutations of the GIP molecule with antagonisticproperties³³⁻³⁹ and recently a potent antagonist antibody against theGIPR⁴⁰.

Under physiological conditions the 42 amino acid hormone, GIP, isdegraded by the enzyme dipeptidylpeptidase 4 (DPP-4), which cleaves atthe third position of the GIP molecule to yield GIP3-42. Syntheticporcine GIP3-42 displayed no antagonist properties in pigs or perfusedrat pancreata in physiological concentrations while in vitro itantagonized the hGIPR⁴¹. Many peptide hormones are post-translationallymodified resulting in various biological forms with different lengthsand amino acid modifications^(42,43). Thus, it has been shown thatGIP1-30 is produced as a result of post-translational processing⁴⁴ andthat it is an agonist on the GIPR^(33,45). If GIP1-30 is secreted intothe circulation in humans, the cleavage catalyzed by DPP-4 would resultin GIP3-30 (see FIG. 1).

U.S. Pat. No. 7,875,587 discloses GIP receptor antagonists derived fromGIP(1-42) having enhanced resistance to degradation by DPP-4, and theiruse for treatment of insulin resistance and obesity. In WO2004/067548DPP-4 metabolites are modified by covalent coupling of a pharmacophoreto achieve the longer half-life associated with the peptide metabolitesand to retain the biological activity of the cleaved peptides similar tothe native peptides, including GIP. WO2012/055770 discloses GIP(3-42) asan endogenous metabolite that is readily cleared and with GIPRantagonist effects, and GIP(2-30) as an example of a truncated GIPanalogue with GIPR agonist activity. WO1998/24464 discloses theantagonist GIP(7-30).

SUMMARY OF INVENTION

The present inventors have characterized GIP peptide analogues andevaluated their affinity to GIPRs, in particular the hGIPR (human GIPR),and their ability to antagonize GIPR activity, in particular the hGIPR.Thus, highly potent antagonists of the hGIPR are disclosed herein.

In one aspect, the invention relates to a peptide consisting of 21 to 39contiguous amino acid residues derived from gastric inhibitory peptide(GIP)

wherein said peptide comprises at least the sequenceTFISDYSIAMDKIX₁QQDFVNW (GIP5-25, SEQ ID NO: 5),

wherein X₁ is any amino acid,

wherein said peptide does not comprise the Tyr amino acid of position 1of SEQ ID NO: 4, and wherein said peptide does not comprise the Alaamino acid of position 2 of SEQ ID NO: 4,

or a functional variant thereof having at least 60% identity to saidpeptide.

In a particular embodiment the peptide of the invention is hGIP3-30 (SEQID NO: 1), hGIP(3-30)H18R or rGIP3-30 (SEQ ID NO: 2), orhGIP(3-30)H18R/K30R or mGIP3-30 (SEQ ID NO: 3), or functional variantsthereof.

In another aspect, the invention relates to the use of such peptides asa medicament.

In yet another aspect, the invention relates to the use of such peptidesin a method of antagonizing a GIP receptor; or treating metabolicdisorders (or metabolic syndrome), such as obesity, diabetes mellitus,insulin resistance and fatty acid metabolism disorder. In other aspectsthe invention relates to methods of treating cancer. In other aspectsthe invention relates to methods of treating a bone density disorder.

DESCRIPTION OF DRAWINGS

FIG. 1. The degradation of hGIP(1-42) and hGIP(1-30) by DPP-4.

FIG. 2. Meal induced hGIP(1-30) response in T2DM patients. PlasmaGIP(1-30) levels were measured from T2DM patients following indigestionof a mixed-meal. Measurements of GIP(1-30) were conducted using aradioimmunoassay with no cross-reactivity with GIP(1-42). Data aremean±SEM, n=10.

FIG. 3. Competition binding with ¹²⁵I-labeled hGIP. The binding of¹²⁵I-labeled hGIP to the transiently transfected COS-7 cells with hGIPRcDNA, was tested in a binding assays vs hGIP(1-42)(▪), GIP(3-30)H18A(•), GIP(3-30)H18R (▾), GIP(3-30)H18K (▪), GIP(3-30)H18R+K30R (♦),hGIP(3-30) (▴). The data was normalized to maximal specific binding andshown as mean±SEM, n≥3.

FIG. 4. Schild plot analysis of GIP(3-30) variants on the hGIPR.hGIP(1-42) induced cAMP accumulation dose-response curves withincreasing concentrations of hGIP(3-30) (A), GIP(3-30)H18R (C), andGIP(3-30)H18R+K30R (E). 0 nM (∘), 17.8 nM (*), 31.6 nM (▾), 56.2 nM (♦),100 nM (•), 178 nM (▪), and 316 nM (▴). The data was normalized to Emaxof each curve and shown as mean±SEM, n≥3. Nonlinear regression was usedto calculate EC50 values. Schild plot analysis of the dose-responsecurves for hGIP(3-30) (B), GIP(3-30)H18R (D), and GIP(3-30)H18R+K30R(F). The x-axis intersect demonstrates a Ki of 15 nM, 14 nM, and 54 nM,respectively.

FIG. 5. Antagonism of hGIP induced somatostatin secretion by hGIP(3-30).Somatostatin secretion following stimulation of perfused rat pancreataby either, 1 nM hGIP, 100 nM hGIP(3-30), a preincubation with 100 nMhGIP(3-30) followed by 1 nM hGIP, or arginine (n=3). The glucoseconcentration was 7 mM and data are mean±SEM.

FIG. 6. GIP(1-30) is a high affinity full agonist of the GIP receptor.Human native GIP(1-42) sequence was acquired from NCBI Protein Database.The GIP receptor was transiently transfected in COS-7 cells and used forfunctional (A) and binding studies (B). A) cAMP accumulation assay withincreased concentrations of native GIP(1-42) (

) and GIP(1-30)NH₂ (n), mean±SEM, n=8. B) Competitive binding with the¹²⁵I-GIP(1-42) radioligand displaced by GIP(1-42) (

) and GIP(1-30) (c), mean±SEM, n=13.

FIG. 7. GIP(3-30)NH₂ and GIP(5-30)NH₂ display highest affinity among theeight truncated GIP variants. The binding of ¹²⁵I-GIP(1-42) totransiently transfected COS-7 cells with the GIP receptor was tested inthe presence of increasing amounts of GIP(1-30)NH₂ (- - - ),GIP(3-30)NH₂ (▪), GIP(5-30)NH₂ (- - - ), GIP(2-30)NH₂ (•), GIP(4-30)NH₂(⋄), GIP(7-30)NH₂ (▴), GIP(8-30)NH₂ (▴), GIP(9-30)NH₂ (Δ), orGIP(6-30)NH₂ (▾), mean±SEM, n=3-13.

FIG. 8. GIP(3-30) and GIP(5-30) are the most potent GIP receptorantagonists. cAMP accumulation in transiently transfected COS-7 cellswith GIP receptor was assessed following incubation with GIP(1-30)NH₂(- - - ), GIP(2-30)NH₂ (•), GIP(3-30)NH₂ (▪), GIP(4-30)NH₂ (⋄),GIP(5-30)NH₂ (∘), GIP(6-30)NH₂ (▾), GIP(7-30)NH₂ (♦), GIP(8-30)NH₂ (▴),or GIP(9-30)NH₂ (Δ). A/B) Ligand dose-response stimulated cAMPaccumulation, mean±SEM, n=3-6. C/D) Dose response curves of antagonistsinhibited a constant amount of native GIP(1-42) corresponding to 50-80%of max receptor activation, mean±SEM, n=4-7.

FIG. 9. Of the six antagonists only GIP(3-30)NH₂ and GIP(5-30)NH₂ arecompetitive antagonists. GIP(1-42) mediated cAMP accumulation assayedfor transiently transfected COS-7 cells with the GIP receptor in theabsence of and with increasing concentrations of either GIP(2-30)NH₂,GIP(3-30)NH₂, GIP(4-30)NH₂, GIP(5-30)NH₂, GIP(6-30)NH₂, or GIP(7-30)NH₂.The corresponding Schild plot is presented with a comparison to a linearregression with a slope of 1.0 and the X-intercept of Ki for theantagonist. GIP(2-30)NH₂ (A), GIP(3-30)NH₂ (B), GIP(4-30)NH₂ (C),GIP(5-30)NH₂ (D), GIP(6-30)NH₂ (E), and GIP(7-30)NH₂ (F), mean±SEM,n=3-6. Antagonist concentrations: 10 nM (▪), 31.6 nM (•), 100 nM (▾),316 nM (♦), 1 μM (▴).

FIG. 10. The homologous binding curves are equivalent to theheterologous binding studies with native ¹²⁵I-GIP(1-42) radioligand.A-C) Transiently transfected COS-7 cells with the GIP receptor were usedin homolog competitive binding studies with ¹²⁵I-GIP(1-30)NH₂ (□),¹²⁵I-GIP(2-30)NH₂ (•), and ¹²⁵I-GIP(3-30)NH₂ (▪) and heterologousbinding studies with ¹²⁵I-GIP(1-42) (- - - ), mean±SEM, n=3-5. D)B_(max)-values calculated from the homologous binding curves forGIP(1-42), GIP(1-30)NH₂, GIP(2-30)NH₂, and GIP(3-30)NH₂, mean±SEM, n=5.

FIG. 11. Human GIP(3-42) is a low potent antagonist on the human GIPreceptor compared to human GIP(3-30) and porcine GIP(3-42). Human andporcine GIP(1-42) sequence was acquired from NCBI Protein Database(UniProtKB-P01281 (GIP_PIG). The human GIP receptor transientlytransfected in COS-7 cells was used in cAMP accumulation assay. A)Dose-response curves of antagonists inhibited a constant amount ofnative GIP(1-42) corresponding to 50-80% of max receptor activation,hGIP(3-42) (o), hGIP(3-30)NH₂ (▪), and pGIP(3-42) (v), mean±SEM, n=3-17.B) Fold change in potency of human GIP(1-42) by 1 μM antagonist. Thebars display mean fold change±SEM, n=3-4.

FIG. 12. Correlation of affinity and antagonistic potency (A) andstructure of the N-terminus of GIP (B). A) The correlation of calculatedaffinities (binding log.IC₅₀) and antagonistic potencies (cAMP log.IC₅₀)plotted for the eight GIP receptor antagonists. GIP(2-30)NH₂ (•),GIP(3-30)NH₂ (▪), GIP(4-30)NH₂ (⋄), GIP(5-30)NH₂ (∘), GIP(6-30)NH₂ (v),GIP(7-30)NH₂ (*), GIP(8-30)NH₂ (A), and GIP(9-30)NH₂ (A). B) Thepublished structure (Parthier et al., 2007) of the native GIP(1-42)peptide with amino acids 1-9 in blue, Glu-3 and Thr-5 in green and Tyr-1and Phe-6 in pink.

FIG. 13: cAMP accumulation assay showing no GIP(3-30) inducedactivation. 35.000 COS-7 cells/well were transiently transfected withhGIPR and stimulated with either GIP(1-42) or hGIP(3-30); n=4.

FIG. 14: Schild plot analysis, using the cAMP accumulation assay,defines GIP(3-30) as a competitive antagonist. 35.000 COS-7 cells/wellwere transiently transfected with hGIPR. A) cAMP production was measuredas a function of GIP concentration in the absence or presence ofincreasing GIP(3-30) concentrations. These schild curves clearlyindicate a competitive nature of GIP(3-30) as seen in the shift inpotency. B) The Schild plot analysis for the dose-response curvesclearly shows the competitive nature of hGIP(3-30), seen as in thelinearity of the plot with a Hill slope of 1.1 and the Ki (X-intercept)of 15 nM.

FIG. 15: Schild plot analysis, using the cAMP accumulation assay,analyses the mutated GIP(3-30) variants as competitive antagonists. Onlyrat GIP(3-30) shows competitive antagonism. 35.000 COS-7 cells/well weretransiently transfected with hGIPR. These schild curves clearly indicateantagonistic properties of the GIP(3-30) variants as seen in the shiftin potency.

DEFINITIONS

The term “affinity” refers to the strength of binding between a receptorand its ligand(s). In the present context, affinity of an antagonist forits binding site (Ki) will determine the duration of inhibition ofagonist activity. The affinity of an antagonist can be determinedexperimentally using Schild regression or for competitive antagonists inradioligand binding studies using the Cheng-Prusoff equation (cf.examples).

The term “IC50” represents the half maximal inhibitory concentration(IC50), which is a measure of the effectiveness of a substance ininhibiting a specific biological or biochemical function. Thisquantitative measure indicates how much of a particular drug or othersubstance (e.g. antagonist) is needed to inhibit a given biologicalprocess (or component of a process, i.e. an enzyme, cell, cell receptoror microorganism) by half. It is commonly used as a measure ofantagonist drug potency in pharmacological research. IC50 represents theconcentration of a drug that is required for 50% inhibition in vitro. Inthe present context, the IC50 value can also refer to the concentrationof a drug at which 50% of a radio labelled ligand is displaced from thereceptor, which is a characterization of drug affinity done incompetition binding experiments.

The term “agonist” in the present context refers to a peptide as definedherein, capable of binding to and activating a receptor.

The term “antagonist” in the present context refers to a peptide asdefined herein, capable of binding to and blocking or reducingagonist-mediated responses of a receptor. Antagonists usually do notprovoke a biological response themselves upon binding to a receptor.Antagonists have affinity but no efficacy for their cognate receptors,and binding will disrupt the interaction and inhibit the function of anagonist or inverse agonist at receptors. Antagonists mediate theireffects by binding to the active (orthosteric) site or to allostericsites on receptors, or they may interact at unique binding sites notnormally involved in the biological regulation of the receptor'sactivity. Antagonist activity may be reversible or irreversibledepending on the longevity of the antagonist-receptor complex, which, inturn, depends on the nature of antagonist-receptor binding. The majorityof drug antagonists typically achieve their potency by competing withendogenous ligands or substrates at structurally defined binding siteson receptors. Antagonists may be competitive, non-competitive,uncompetitive, silent antagonists, partial agonists or inverse agonists.

A competitive antagonist (also known as surmountable antagonist)reversibly binds to receptors at the same binding site (i.e. at theactive site) as the endogenous ligand or agonist, but without activatingthe receptor. Agonists and antagonists thus “compete” for the samebinding site on the receptor. Once bound, an antagonist blocks agonistbinding. The level of activity of the receptor is determined by therelative affinity of each molecule for the site and their relativeconcentrations. High concentrations of a competitive antagonist willincrease the proportion of receptors that the antagonist occupies;higher concentrations of the agonist will be required to obtain the samedegree of binding site occupancy.

The term “non-competitive antagonism” (also called nonsurmountable orinsurmountable antagonism) describes two distinct phenomena withfunctionally similar results: one in which the antagonist binds to theactive site of the receptor, and one in which the antagonist binds to anallosteric site of the receptor. Unlike competitive antagonists, whichaffect the amount of agonist necessary to achieve a maximal response butdo not affect the magnitude of that maximal response, non-competitiveantagonists reduce the magnitude of the maximum response that can beattained by any amount of agonist.

An uncompetitive antagonist requires receptor activation by an agonistbefore it can bind to a separate allosteric binding site. This type ofantagonism produces a kinetic profile in which the same amount ofantagonist blocks higher concentrations of agonist better than lowerconcentrations of agonist.

The term “silent antagonist” refers to a competitive receptor antagonistthat has absolutely no intrinsic activity for activating a receptor.

The term “partial agonist” refers to an agonist that, at a givenreceptor, might differ in the amplitude of the functional response thatit elicits after maximal receptor occupancy. Partial agonists can act asa competitive antagonist in the presence of a full agonist (or a moreefficacious agonist), as it competes with the full agonist for receptoroccupancy, thereby producing a net decrease in the receptor activationas compared to that observed with the full agonist alone.

The term “inverse agonist” refers to agonists having effects similar tothose of antagonists, but causing a distinct set of downstreambiological responses. Constitutively active receptors that exhibitintrinsic or basal activity can have inverse agonists, which not onlyblock the effects of binding agonists like a classical antagonist butalso inhibit the basal activity of the receptor.

The term “Individual” refers to vertebrates, particular members of themammalian species, preferably primates including humans. As used herein,‘subject’ and ‘individual’ may be used interchangeably.

An “isolated peptide” is a peptide separated and/or recovered from acomponent of their natural, typically cellular, environment, that isessentially free from contaminating cellular components, such ascarbohydrate, lipid, or other proteinaceous impurities associated withthe polypeptide in nature. Typically, a preparation of isolated peptidecontains the peptide in a highly purified form, i.e., at least about 80%pure, at least about 90% pure, at least about 95% pure, greater than 95%pure, or greater than 99% pure. The term “isolated” does not exclude thepresence of the same peptide in alternative physical forms, such asdimers, tetramers or alternatively glycosylated or derived forms.

An “amino acid residue” can be a natural or non-natural amino acidresidue linked by peptide bonds or bonds different from peptide bonds.The amino acid residues can be in D-configuration or L-configuration. Anamino acid residue comprises an amino terminal part (NH₂) and a carboxyterminal part (COOH) separated by a central part comprising a carbonatom, or a chain of carbon atoms, at least one of which comprises atleast one side chain or functional group. NH₂ refers to the amino grouppresent at the amino terminal end of an amino acid or peptide, and COOHrefers to the carboxy group present at the carboxy terminal end of anamino acid or peptide. The generic term amino acid comprises bothnatural and non-natural amino acids. Natural amino acids of standardnomenclature as listed in J. Biol. Chem., 243:3552-59 (1969) and adoptedin 37 C.F.R., section 1.822(b)(2) belong to the group of amino acidslisted herewith: Y,G,F,M,A,S,I,L,T,V,P,K,H,Q,E,W,R,D,N and C.Non-natural amino acids are those not listed immediately above. Also,non-natural amino acid residues include, but are not limited to,modified amino acid residues, L-amino acid residues, and stereoisomersof D-amino acid residues.

An “equivalent amino acid residue” refers to an amino acid residuecapable of replacing another amino acid residue in a polypeptide withoutsubstantially altering the structure and/or functionality of thepolypeptide. Equivalent amino acids thus have similar properties such asbulkiness of the side-chain, side chain polarity (polar or non-polar),hydrophobicity (hydrophobic or hydrophilic), pH (acidic, neutral orbasic) and side chain organization of carbon molecules(aromatic/aliphatic). As such, “equivalent amino acid residues” can beregarded as “conservative amino acid substitutions”.

Within the meaning of the term “equivalent amino acid substitution” asapplied herein, one amino acid may be substituted for another, in oneembodiment, within the groups of amino acids indicated herein below:

-   -   i) Amino acids having polar side chains (Asp, Glu, Lys, Arg,        His, Asn, Gln, Ser, Thr, Tyr, and Cys)    -   ii) Amino acids having non-polar side chains (Gly, Ala, Val,        Leu, lie, Phe, Trp, Pro, and Met)    -   iii) Amino acids having aliphatic side chains (Gly, Ala Val,        Leu, lie)    -   iv) Amino acids having cyclic side chains (Phe, Tyr, Trp, His,        Pro)    -   v) Amino acids having aromatic side chains (Phe, Tyr, Trp)    -   vi) Amino acids having acidic side chains (Asp, Glu)    -   vii) Amino acids having basic side chains (Lys, Arg, His)    -   viii) Amino acids having amide side chains (Asn, Gln)    -   ix) Amino acids having hydroxy side chains (Ser, Thr)    -   x) Amino acids having sulphur-containing side chains (Cys, Met),    -   xi) Neutral, weakly hydrophobic amino acids (Pro, Ala, Gly, Ser,        Thr)    -   xii) Hydrophilic, acidic amino acids (Gln, Asn, Glu, Asp), and    -   xiii) Hydrophobic amino acids (Leu, lie, Val)

Where the L or D form (optical isomers) has not been specified it is tobe understood that the amino acid in question has the natural L form,cf. Pure & Appl. Chem. Vol. (56(5) pp 595-624 (1984) or the D form, sothat the peptides formed may be constituted of amino acids of L form, Dform, or a sequence of mixed L forms and D forms.

A “functional variant” of a peptide is a peptide capable of performingessentially the same functions as the peptide it is a functional variantof. In particular, a functional variant can bind the same molecules,preferably with the same affinity, as the peptide it is a functionalvariant of.

A “bioactive agent” (i.e. a biologically active substance/agent) is anyagent, drug, compound, composition of matter or mixture which providessome pharmacologic, often beneficial, effect that can be demonstrated invivo or in vitro. It refers to the peptide sequences according to thepresent invention, compounds or compositions comprising these andnucleic acid constructs encoding said peptides. As used herein, thisterm further includes any physiologically or pharmacologically activesubstance that produces a localized or systemic effect in an individual.A ‘bioactive agent’ as used herein denotes collectively a peptide, anucleic acid construct encoding said peptide, and a compositioncomprising a peptide according to the present invention.

The terms “drug” and “medicament” as used herein include biologically,physiologically, or pharmacologically active substances that act locallyor systemically in the human or animal body.

The terms “treatment” and “treating” as used herein refer to themanagement and care of a patient for the purpose of combating acondition, disease or disorder. The term is intended to include the fullspectrum of treatments for a given condition from which the patient issuffering, and refer equally to curative therapy, prophylactic orpreventative therapy and ameliorating or palliative therapy, such asadministration of the peptide or composition for the purpose of:alleviating or relieving symptoms or complications; delaying theprogression of the condition, partially arresting the clinicalmanifestations, disease or disorder; curing or eliminating thecondition, disease or disorder; amelioration or palliation of thecondition or symptoms, and remission (whether partial or total), whetherdetectable or undetectable; and/or preventing or reducing the risk ofacquiring the condition, disease or disorder, wherein “preventing” or“prevention” is to be understood to refer to the management and care ofa patient for the purpose of hindering the development of the condition,disease or disorder, and includes the administration of the activecompounds to prevent or reduce the risk of the onset of symptoms orcomplications. The term “palliation”, and variations thereof, as usedherein, means that the extent and/or undesirable manifestations of aphysiological condition or symptom are lessened and/or time course ofthe progression is slowed or lengthened, as compared to notadministering compositions of the present invention.

The individual to be treated is preferably a mammal, in particular ahuman being. Treatment of animals, such as mice, rats, dogs, cats, cows,horses, sheep and pigs, is, however, also within the scope of thepresent invention.

An “individual in need thereof” refers to an individual who may benefitfrom the present invention. In one embodiment, said individual in needthereof is a diseased individual, wherein said disease may be ametabolic disease or disorder such as obesity or diabetes, a bonedensity disorder or a cancer.

A “treatment effect” or “therapeutic effect” is manifested if there is achange in the condition being treated, as measured by the criteriaconstituting the definition of the terms “treating” and “treatment.”There is a “change” in the condition being treated if there is at least5% improvement, preferably 10% improvement, more preferably at least25%, even more preferably at least 50%, such as at least 75%, and mostpreferably at least 100% improvement. The change can be based onimprovements in the severity of the treated condition in an individual,or on a difference in the frequency of improved conditions inpopulations of individuals with and without treatment with the bioactiveagent, or with the bioactive agent in combination with a pharmaceuticalcomposition of the present invention.

A treatment according to the invention can be prophylactic, amelioratingand/or curative.

“Pharmacologically effective amount”, “pharmaceutically effectiveamount” or “physiologically effective amount” of a “bioactive agent” isthe amount of a bioactive agent present in a pharmaceutical compositionas described herein that is needed to provide a desired level of activeagent in the bloodstream or at the site of action in an individual (e.g.the lungs, the gastric system, the colorectal system, prostate, etc.) tobe treated to give an anticipated physiological response when suchcomposition is administered.

“Co-administering” or “co-administration” as used herein refers to theadministration of one or more peptides of the present invention and astate-of-the-art pharmaceutical composition. The at least two componentscan be administered separately, sequentially or simultaneously.

DETAILED DESCRIPTION OF THE INVENTION

GIP refers to glucose-dependent insulinotropic polypeptide, also knownas Gastric Inhibitory Peptide (or polypeptide). As used herein theabbreviation hGIP is human GIP (Uniprot accession number P09681), mGIPis mouse GIP (Uniprot accession number P48756) and rGIP is rat GIP(Uniprot accession number Q06145). GIP is derived from a 153-amino acidproprotein and circulates as a biologically active 42-amino acidpeptide. It is synthesized by K cells of the mucosa of the duodenum andthe jejunum of the gastrointestinal tract.

GIPR (or GIP receptor) refers to gastric inhibitory polypeptidereceptors. These seven-transmembrane proteins are found at least onbeta-cells in the pancreas. As used herein the abbreviation hGIPR ishuman GIPR (Uniprot accession number P48546), mGIPR is mouse GIPR(Uniprot accession number Q0P543) and rGIPR is rat GIPR (Uniprotaccession number P43219).

The present inventors have identified GIP analogues with novelproperties and surprisingly found that the GIP analogues of the presentinvention are antagonists of one or more GIPRs. This makes thempotentially useful in a range of therapeutic applications.

Peptides According to the Invention

The present invention is directed to GIP peptide analogues which GIPpeptide analogues do not comprise the two most N-terminal amino acidresidues of native GIP. In some embodiments the GIP peptide analogues donot comprise the 2, 3 or 4 most N-terminal amino acid residues of nativeGIP.

It is an aspect of the invention to provide a peptide consisting of 21to 39 contiguous amino acid residues derived from gastric inhibitorypeptide (GIP) (e.g. SEQ ID NO: 4), wherein said peptide comprises atleast the sequence

(GIP5-25; SEQ ID NO: 73) TFISDYSIAMX _(0a) X _(0b)IX ₁QQDFVNW or(GIP5-25, SEQ ID NO: 5) TFISDYSIAMDKIX ₁QQDFVNW,

wherein X_(0a), X_(0b), X₁ and X₁ individually is any amino acid,

wherein said peptide does not comprise the Tyr amino acid of position 1of SEQ ID NO: 4 (nor any other amino acid at position 1 of SEQ ID NO:4), and wherein said peptide does not comprise the Ala amino acid ofposition 2 of SEQ ID NO: 4 (nor any other amino acid at position 2 ofSEQ ID NO: 4),

or a functional variant thereof having at least 60% identity to saidpeptide.

In one embodiment the peptides are selected from the group consisting of

(GIP3-30; SEQ ID NO: 74) EGTFISDYSIAMX _(0a) X _(0b)IX ₁QQDFVNWLLAQX ₂,(GIP4-30; SEQ ID NO: 75) GTFISDYSIAMX _(0a) X _(0b)IX ₁QQDFVNWLLAQX ₂and (GIP5-30; SEQ ID NO: 76) TFISDYSIAMX _(0a) X _(0b)IX ₁QQDFVNWLLAQX₂,

wherein X_(0a), X_(0b), X₁ and X₂ are individually any amino acid.

In one embodiment the peptides are selected from any one of SEQ ID NOs:1-3, 5-61 and 72-91.

In one embodiment the peptides are selected from the group consisting of

EGTFISDYSIAMX _(0a) X _(0b)IX ₁QQDFVNWLLAQX ₂-NH₂, GTFISDYSIAMX _(0a) X_(0b)IX ₁QQDFVNWLLAQX ₂-NH₂ and TFISDYSIAMX _(0a) X _(0b)IX₁QQDFVNWLLAQX ₂-NH₂,wherein X_(0a), X_(0b), X₁ and X₂ are individually any amino acid.

It is a further aspect of the invention to provide hGIP(2-30),hGIP(6-30), hGIP(7-30), hGIP(8-30) and hGIP(9-30), or a functionalvariant thereof, per se or in a method as defined herein elsewhere.

In one embodiment, the peptides of the invention are capable of bindingto and antagonising a GIPR. In some embodiments, the GIPR is the humanGIPR (Uniprot accession number P48546), the mouse GIPR (Uniprotaccession number Q0P543) or the rat GIPR (Uniprot accession numberP43219).

The terms ‘peptide’ and ‘isolated peptide’ may be used interchangeablyherein. The terms ‘variant’ and ‘functional variant’ may be usedinterchangeably herein. According to the present invention, a peptide asdefined herein may be a functional variant of said defined amino acidsequence.

When reference is made to a ‘peptide’ herewith, this term will encompassboth references to a peptide per se, and also to a peptide for useaccording to the present invention.

Functional variants of the peptides according to the present inventionare the functional equivalents of said sequences, i.e. they retain atleast some effect associated with the native sequence.

In one embodiment a functional variant retains the same biologicalactivity or capabilities as the native peptide or the peptide from whichit is derived. In one embodiment a peptide and a functional variantthereof according to the present invention is capable of one or more of:Binding to one or more GIPRs; antagonizing one or more GIPRs; displacingGIP1-42 and/or GIP1-30 from one or more GIPRs; having a higher affinityfor a given GIPR than GIP1-42 and/or GIP1-30; antagonizing somatostatinsecretion induced by native GIP, GIP1-42 and/or GIP1-30; antagonizinginsulin secretion induced by native GIP, GIP1-42 and/or GIP1-30; andantagonising glucagon secretion induced by native GIP, GIP1-42 and/orGIP1-30.

In one embodiment a peptide and a functional variant thereof accordingto the invention is capable of binding (or binds) to one or more of thehGIPR (Uniprot accession number P48546), the rGIPR (Uniprot accessionnumber P43219) and the mGIPR (Uniprot accession number Q0P543).

In one embodiment a peptide and a functional variant thereof accordingto the invention is capable of inhibiting (reducing, antagonizing) oneor more of i) GIP-induced glucagon secretion, ii) GIP-induced insulinsecretion, iii) GIP-induced somatostatin secretion, iv) GIP-inducedglucose uptake, v) GIP-induced fatty acid synthesis and/or fatty acidincorporation, vi) high or increased expression or activity of a GIPRand vii) release of GIP following a meal (post-prandial GIP release).

In one embodiments, the peptide comprises at least the sequenceTFISDYSIAMDKIX₁QQDFVNW (GIP5-25, SEQ ID NO: 5), wherein X₁ at position18 is any amino acid, such as a naturally occurring or a non-naturallyoccurring amino acid as defined herein.

In one embodiments, the peptide comprises at least the sequenceTFISDYSIAMDKIX₁QQDFVNWLLAQX₂ (GIP5-30, SEQ ID NO: 78), wherein X₁ atposition 18 and X₂ at position 30 is individually any amino acid, suchas a naturally occurring or a non-naturally occurring amino acid asdefined herein.

In one embodiment, the peptides of the invention do not comprise thefirst two amino acids of GIP, i.e. the peptides of the invention do notcomprise the Tyr amino acid of position 1 and do not comprise the Alaamino acid of position 2 of SEQ ID NO: 4.

A peptide that comprises or consists of a sequence means that thepeptide can comprise the sequence, consist of the sequence, or compriseat least the full sequence. A peptide that ‘comprises at least’ apeptide sequence, such as ‘comprising at least the sequenceTFISDYSIAMDKIX₁QQDFVNW’, means that the peptide includes all of thepeptide sequence TFISDYSIAMDKIX₁QQDFVNW (GIP5-25, SEQ ID NO: 5).

It does, however, not exclude that additional components or amino acidsare present.

In one embodiment the peptide is non-naturally occurring.

In one embodiment the peptide is synthetic.

In one embodiment the peptide is an isolated peptide.

In one embodiment the peptide is selected from the group consisting of

(GIP3-30; SEQ ID NO: 74) EGTFISDYSIAMX _(0a) X _(0b)IX ₁QQDFVNWLLAQX ₂,(GIP4-30; SEQ ID NO: 75) GTFISDYSIAMX _(0a) X _(0b)IX ₁QQDFVNWLLAQX ₂and (GIP5-30; SEQ ID NO: 76) TFISDYSIAMX _(0a) X _(0b)IX ₁QQDFVNWLLAQX₂,

wherein X_(0a), X_(0b), X₁ and X₂ are individually any amino acid,

or a functional variant thereof having at least 75% sequence identity tosaid peptide.

In one embodiment the peptide is selected from the group consisting of

(GIP3-30; SEQ ID NO: 11) EGTFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂,(GIP4-30; SEQ ID NO: 28) GTFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂,(GIP5-30; SEQ ID NO: 45) TFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂,

wherein X_(0a), X_(0b), X₁ and X₂ are individually any amino acid,

or a functional variant thereof having at least 75% sequence identity tosaid peptide.

The peptide of the invention is in one embodiment selected from thegroup consisting of:

(GIP3-25, SEQ ID NO: 6) EGTFISDYSIAMDKIX ₁QQDFVNW,(GIP3-26, SEQ ID NO: 7) EGTFISDYSIAMDKIX ₁QQDFVNWL,(GIP3-27, SEQ ID NO: 8) EGTFISDYSIAMDKIX ₁QQDFVNWLL,(GIP3-28, SEQ ID NO: 9) EGTFISDYSIAMDKIX ₁QQDFVNWLLA,(GIP3-29, SEQ ID NO: 10) EGTFISDYSIAMDKIX ₁QQDFVNWLLAQ,(GIP3-30, SEQ ID NO: 11) EGTFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂,(GIP3-31, SEQ ID NO: 12) EGTFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂G,(GIP3-32, SEQ ID NO: 13) EGTFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂GK,(GIP3-33, SEQ ID NO: 14) EGTFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂GKK,(GIP3-34, SEQ ID NO: 15) EGTFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂GKKN,(GIP3-35, SEQ ID NO: 16) EGTFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂GKKND,(GIP3-36, SEQ ID NO: 17) EGTFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂GKKNDW,(GIP3-37, SEQ ID NO: 18) EGTFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂GKKNDWK,(GIP3-38, SEQ ID NO: 19) EGTFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂GKKNDWKH,(GIP3-39, SEQ ID NO: 20) EGTFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂GKKNDWKHN,(GIP3-40, SEQ ID NO: 21) EGTFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂GKKNDWKHNI,(GIP3-41, SEQ ID NO: 22) EGTFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂GKKNDWKHNIT,

or a functional variant thereof, wherein X₁ and X₂ are individually anyamino acid, such as a naturally occurring or a non-naturally occurringamino acid as defined herein.

In a particular embodiment the peptide of the invention isEGTFISDYSIAMDKIX₁QQDFVNWLLAQX₂ (GIP3-30, SEQ ID NO: 11), or a functionalvariant thereof, wherein X₁ and X₂ are individually any amino acid.

In a particular embodiment, the peptide is hGIP3-30 (SEQ ID NO: 1), or avariant thereof. In a preferred embodiment, the peptide consists ofhGIP3-30 (SEQ ID NO: 1).

In a particular embodiment, the peptide is rGIP3-30 (SEQ ID NO: 2), or avariant thereof. In a preferred embodiment, the peptide consists ofrGIP3-30 (SEQ ID NO: 2).

In a particular embodiment, the peptide is mGIP3-30 (SEQ ID NO: 3), or avariant thereof. In a preferred embodiment, the peptide consists ofmGIP3-30 (SEQ ID NO: 3).

In some embodiment the peptide of the invention does not comprise theGlu amino acid of position 3 (or any other amino acid at position 3) ofSEQ ID NO: 4. In this embodiment the peptide is selected from the groupconsisting of:

(GIP4-25, SEQ ID NO: 23) GTFISDYSIAMDKIX ₁QQDFVNW,(GIP4-26, SEQ ID NO: 24) GTFISDYSIAMDKIX ₁QQDFVNWL,(GIP4-27, SEQ ID NO: 25) GTFISDYSIAMDKIX ₁QQDFVNWLL,(GIP4-28, SEQ ID NO: 26) GTFISDYSIAMDKIX ₁QQDFVNWLLA,(GIP4-29, SEQ ID NO: 27) GTFISDYSIAMDKIX ₁QQDFVNWLLAQ,(GIP4-30, SEQ ID NO: 28) GTFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂,(GIP4-31, SEQ ID NO: 29) GTFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂G,(GIP4-32, SEQ ID NO: 30) GTFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂GK,(GIP4-33, SEQ ID NO: 31) GTFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂GKK,(GIP4-34, SEQ ID NO: 32) GTFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂GKKN,(GIP4-35, SEQ ID NO: 33) GTFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂GKKND,(GIP4-36, SEQ ID NO: 34) GTFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂GKKNDW,(GIP4-37, SEQ ID NO: 35) GTFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂GKKNDWK,(GIP4-38, SEQ ID NO: 36) GTFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂GKKNDWKH,(GIP4-39, SEQ ID NO: 37) GTFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂GKKNDWKHN,(GIP4-40, SEQ ID NO: 38) GTFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂GKKNDWKHNI,(GIP4-41, SEQ ID NO: 39) GTFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂GKKNDWKHNIT,(GIP4-42, SEQ ID NO: 40) GTFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂GKKNDWKHNITQ,

or a functional variant thereof, wherein X₁ and X₂ are individually anyamino acid, such as a naturally occurring or a non-naturally occurringamino acid as defined herein.

In some embodiment the peptide of the invention does not comprise theGlu amino acid of position 3 (or any other amino acid at position 3) andthe Gly amino acid of position 4 (or any other amino acid at position 4)of SEQ ID NO: 4. In this embodiment the peptide is selected from thegroup consisting of:

(GIP5-25, SEQ ID NO: 5) TFISDYSIAMDKIX ₁QQDFVNW,(GIP5-26, SEQ ID NO: 41) TFISDYSIAMDKIX ₁QQDFVNWL,(GIP5-27, SEQ ID NO: 42) TFISDYSIAMDKIX ₁QQDFVNWLL,(GIP5-28, SEQ ID NO: 43) TFISDYSIAMDKIX ₁QQDFVNWLLA,(GIP5-29, SEQ ID NO: 44) TFISDYSIAMDKIX ₁QQDFVNWLLAQ,(GIP5-30, SEQ ID NO: 45) TFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂,(GIP5-31, SEQ ID NO: 46) TFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂G,(GIP5-32, SEQ ID NO: 47) TFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂GK,(GIP5-33, SEQ ID NO: 48) TFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂GKK,(GIP5-34, SEQ ID NO: 49) TFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂GKKN,(GIP5-35, SEQ ID NO: 50) TFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂GKKND,(GIP5-36, SEQ ID NO: 51) TFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂GKKNDW,(GIP5-37, SEQ ID NO: 52) TFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂GKKNDWK,(GIP5-38, SEQ ID NO: 53) TFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂GKKNDWKH(GIP5-39, SEQ ID NO: 54) TFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂GKKNDWKHN,(GIP5-40, SEQ ID NO: 55) TFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂GKKNDWKHNI,(GIP5-41, SEQ ID NO: 56) TFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂GKKNDWKHNIT,(GIP5-42, SEQ ID NO: 57) TFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂GKKNDWKHNITQ,

or a functional variant thereof, wherein X₁ and X₂ are individually anyamino acid, such as a naturally occurring or a non-naturally occurringamino acid as defined herein.

In one embodiment X₁ and X₂ as defined herein for peptide variants areidentical to the amino acid in the corresponding position in human GIP.

In one embodiment X₁ is His and/or X₂ is Lys.

In one embodiment X₁ and X₂ as defined herein for peptide variants areidentical to the amino acid in the corresponding position in rat and/ormouse GIP.

In one embodiment X₁ is Arg and/or X₂ is Lys.

In one embodiment X₁ is Arg and/or X₂ is Arg.

In one embodiment X₁ (position 18) is any amino acid, such as anaturally occurring or a non-naturally occurring amino acid. Inpreferred embodiments, X₁ is selected from the group consisting of Ala,His, Arg and Lys. In these embodiments, the peptides of the inventionconsist of 21 to 39 contiguous amino acid residues and comprises orconsists of at least a sequence selected from the group consisting of

(SEQ ID NO: 59) TFISDYSIAMDKIHQQDFVNW, (SEQ ID NO: 60)TFISDYSIAMDKIRQQDFVNW, (SEQ ID NO: 61) TFISDYSIAMDKIAQQDFVNW and(SEQ ID NO: 72) TFISDYSIAMDKIKQQDFVNW.

In one embodiment X₂ (position 30) is any amino acid, such as anaturally occurring or a non-naturally occurring amino acid. Inpreferred embodiments, X₂ is selected from the group consisting of Ala,Lys and Arg.

In one embodiment X₁ is His. In one embodiment X₁ is His and X₂ is Lys.In another embodiment X₁ is His and X₂ is Arg. In another embodiment X₁is His and X₂ is Ala.

In one embodiment X₁ is Arg. In another embodiment X₁ is Arg and X₂ isLys. In another embodiment X₁ is Arg and X₂ is Arg. In anotherembodiment X₁ is Arg and X₂ is Ala.

In another embodiment X₁ is Ala and X₂ is Lys. In another embodiment X₁is Ala and X₂ is Arg. In another embodiment X₁ is Ala and X₂ is Ala.

In another embodiment X₁ is Lys and X₂ is Lys. In another embodiment X₁is Lys and X₂ is Arg. In another embodiment X₁ is Lys and X₂ is Ala.

In one embodiment the peptide of the invention comprises the sequenceTFISDYSIAMDK IX₁QQDFVNW (GIP5-25, SEQ ID NO: 5), or a variant thereofwhich is TFISDYSIAMX_(0a)X_(0b)IX₁QQDFVNW, wherein D at position 15(X_(0a)) and/or K at position 16 (X_(0b)) are individually any aminoacid, such as a naturally occurring or a non-naturally occurring aminoacid as defined herein.

In one embodiment X_(0a) is selected from Asp and Ala. In one embodimentX_(0b) is selected from Lys and Ala.

It is an aspect of the invention to provide a peptide consisting of 28contiguous amino acid residues derived from gastric inhibitory peptide,wherein said peptide comprises or consists of a peptide selected fromthe group consisting of

(hGIP3-30, SEQ ID NO: 1) EGTFISDYSIAMDKIHQQDFVNWLLAQK,(rGIP3-30, SEQ ID NO: 2) EGTFISDYSIAMDKIRQQDFVNWLLAQK,(mGIP3-30, SEQ ID NO: 3) EGTFISDYSIAMDKIRQQDFVNWLLAQR,

and functional variants thereof having at least 60% identity to saidpeptide.

‘Identity’ and ‘sequence identity’ is used interchangeably herein.

In one embodiment a functional variant according to the presentinvention is selected from the group consisting of:

(hGIP(3-30)H18A; SEQ ID NO: 79) EGTFISDYSIAMDKIAQQDFVNWLLAQK,(hGIP(3-30)H18K; SEQ ID NO: 80) EGTFISDYSIAMDKIKQQDFVNWLLAQK,(hGIP(3-30)D15EH18A; SEQ ID NO: 81) EGTFISDYSIAMEKIAQQDFVNWLLAQK,(hGIP(3-30)K16AH18A; SEQ ID NO: 82) EGTFISDYSIAMDAIAQQDFVNWLLAQK,(hGIP(3-30)D15E; SEQ ID NO: 83) EGTFISDYSIAMEKIHQQDFVNWLLAQK,(hGIP(3-30)D15N; SEQ ID NO: 84) EGTFISDYSIAMNKIHQQDFVNWLLAQK,(hGIP(3-30)K16A; SEQ ID NO: 85) EGTFISDYSIAMDAIHQQDFVNWLLAQK,(hGIP(3-30)K16H; SEQ ID NO: 86) EGTFISDYSIAMDHIHQQDFVNWLLAQK,(hGIP(3-30)K16R; SEQ ID NO: 87) EGTFISDYSIAMDRIHQQDFVNWLLAQK,(hGIP(3-30)H18F; SEQ ID NO: 88) EGTFISDYSIAMDKIFQQDFVNWLLAQK,(hGIP(3-30)H18W; SEQ ID NO: 89) EGTFISDYSIAMDKIWQQDFVNWLLAQK,(hGIP(3-30)K30R SEQ ID NO: 90) EGTFISDYSIAMDKIHQQDFVNWLLAQR, and(hGIP(3-30)K30H; SEQ ID NO: 91) EGTFISDYSIAMDKIHQQDFVNWLLAQH.

In one embodiment, the peptide of the invention has at least 60%identity, such as at least 65% identity, such as at least 70% identity,such as at least 75% identity, such as at least 80% identity, such as atleast 85% identity, such as at least 90% identity, such as at least 95%identity, such as at least 99% identity, such as 100% identity to thecorresponding part of hGIP (SEQ ID NO: 65), rGIP (SEQ ID NO: 66) or mGIP(SEQ ID NO: 67).

In one embodiment, the peptide of the invention has 60 to 65% identity,such as 65 to 70% identity, such as 70 to 75% identity, such as 75 to80% identity, such as 80 to 85% identity, such as 85 to 90% identity,such as 90 to 95% identity, such as 95 to 99% identity, such as 99 to100% identity, such as 100% identity to the corresponding part of hGIP(SEQ ID NO: 65). In some embodiments, the peptide of the invention has60 to 65% identity, such as 65 to 70% identity, such as 70 to 75%identity, such as 75 to 80% identity, such as 80 to 85% identity, suchas 85 to 90% identity, such as 90 to 95% identity, such as 95 to 99%identity, such as 99 to 100% identity, such as 100% identity to hGIP3-30(SEQ ID NO: 1), hGIP4-30 (SEQ ID NO:77), hGIP5-30 (SEQ ID NO:78),rGIP3-30 (SEQ ID NO: 2) or mGIP3-30 (SEQ ID NO: 3).

In one embodiment a variant of a peptide according to the presentinvention is a variant having 1 to 10 amino acid substitutions, such as1 amino acid substitution, for example 2 amino acid substitutions, suchas 3 amino acid substitutions, for example 4 amino acid substitutions,such as 5 amino acid substitutions, for example 6 amino acidsubstitutions, such as 7 amino acid substitutions, for example 8 aminoacid substitutions, such as 9 amino acid substitutions, for example 10amino acid substitutions as compared to the corresponding part of anyone of SEQ ID NOs: 1 to 91/at a given position of any one of SEQ ID NOs:1 to 91.

In one embodiment, one or more, or all, of said amino acid substitutionsare conservative amino acid substitutions.

In one embodiment, the peptide according to the present invention doesnot comprise or consist of GIP1-42 (SEQ ID NO: 4). It follows that inone embodiment, the peptide according to the present invention does notcomprise or consist of hGIP1-42 (SEQ ID NO: 65), rGIP1-42 (SEQ ID NO:66) or mGIP1-42 (SEQ ID NO: 67).

In one embodiment, the peptide according to the present invention doesnot comprise or consist of the amino acid sequence GIP3-42 (SEQ ID NO:58). It follows that in one embodiment, the peptide according to thepresent invention does not comprise or consist of hGIP3-42 (SEQ ID NO:62), rGIP3-42 (SEQ ID NO: 63) or mGIP3-42 (SEQ ID NO: 64).

In one embodiment, the peptide according to the present invention doesnot comprise or consist of GIP1-30 (SEQ ID NO: 68). It follows that inone embodiment, the peptide according to the present invention does notcomprise or consist of hGIP1-30 (SEQ ID NO: 69), rGIP1-30 (SEQ ID NO:70) or mGIP1-30 (SEQ ID NO: 71).

In some embodiments, said one or more amino acid substitution(s) is aconservative amino acid substitution (or synonymous substitution). Aconservative substitution is the substitution of amino acids whose sidechains have similar biochemical properties and thus do not affect thefunction of the peptide.

Among the common amino acids, for example, a “conservative amino acidsubstitution” can also be illustrated by a substitution among aminoacids within each of the following groups: (1) glycine, alanine, valine,leucine, and isoleucine, (2) phenylalanine, tyrosine, and tryptophan,(3) serine and threonine, (4) aspartate and glutamate, (5) glutamine andasparagine, and (6) lysine, arginine and histidine.

In one embodiment, a serine residue of a peptide of the invention issubstituted with an amino acid selected from the group consisting ofGln, Asn and Thr (all amino acids with polar uncharged side chains); andindependently thereof, a glycine residue (Gly) is substituted with anamino acid selected from the group consisting of Ala, Val, Leu, and lie;and independently thereof, an arginine residue (Arg) is substituted withan amino acid selected from the group consisting of Lys and His (allhave positively charged side chains); and independently thereof, alysine residue (Lys) is substituted with an amino acid selected from thegroup consisting of Arg and His; and independently thereof, a methionineresidue (Met) is substituted with an amino acid selected from the groupconsisting of Leu, Pro, lie, Val, Phe, Tyr and Trp (all have hydrophobicside chains); and independently thereof, a glutamine residue (Gln) issubstituted with an amino acid selected from the group consisting ofAsp, Glu, and Asn; and independently thereof, an alanine residue (Ala)is substituted with an amino acid selected from the group consisting ofGly, Val, Leu, and Ile.

Particular amino acid substitutions of the present invention are K to R,E to D, L to M, Q to E, I to V, I to L, A to S, Y to W, K to Q, S to T,N to S and Q to R.

The identity between amino acid sequences may be calculated using wellknown algorithms such as BLOSUM 30, BLOSUM 40, BLOSUM 45, BLOSUM 50,BLOSUM 55, BLOSUM 60, BLOSUM 62, BLOSUM 65, BLOSUM 70, BLOSUM 75, BLOSUM80, BLOSUM 85, or BLOSUM 90, or by simple comparison of the specificamino acids present at corresponding positions in two peptide sequencesto be compared.

Homology may be used as a synonym to identity/sequence identity.

In another embodiment, a variant according to the present inventionincludes sequences wherein an alkyl amino acid is substituted for analkyl amino acid, wherein an aromatic amino acid is substituted for anaromatic amino acid, wherein a sulfur-containing amino acid issubstituted for a sulfur-containing amino acid, wherein ahydroxy-containing amino acid is substituted for a hydroxy-containingamino acid, wherein an acidic amino acid is substituted for an acidicamino acid, wherein a basic amino acid is substituted for a basic aminoacid, and/or wherein a dibasic monocarboxylic amino acid is substitutedfor a dibasic monocarboxylic amino acid.

Conservative substitutions may be introduced in any one or morepositions of a peptide according to the invention, as long as thevariant remains functional. It may however also be desirable tointroduce non-conservative substitutions in one or more positions(non-synonymous substitutions).

A non-conservative substitution leading to the formation of a variant ofthe peptide according to the invention in one embodiment comprisessubstitution of amino acid residues that i) differ substantially inpolarity, for example a residue with a non-polar side chain (Ala, Leu,Pro, Trp, Val, lie, Leu, Phe or Met) substituted for a residue with apolar side chain such as Gly, Ser, Thr, Cys, Tyr, Asn, or Gln or acharged amino acid such as Asp, Glu, Arg, or Lys, or substituting acharged or a polar residue for a non-polar one; and/or ii) differsubstantially in its effect on peptide backbone orientation such assubstitution of or for Pro or Gly by another residue; and/or iii) differsubstantially in electric charge, for example substitution of anegatively charged residue such as Glu or Asp for a positively chargedresidue such as Lys, His or Arg (and vice versa); and/or iv) differsubstantially in steric bulk, for example substitution of a bulkyresidue such as His, Trp, Phe or Tyr for one having a minor side chain,e.g. Ala, Gly or Ser (and vice versa).

Substitution of amino acids can in one embodiment be made based upontheir hydrophobicity and hydrophilicity values and the relativesimilarity of the amino acid side-chain substituents, including charge,size, and the like.

The peptides according to the present invention comprise proteinogenicor natural amino acids, i.e. the 22 amino acids naturally incorporatedinto polypeptides. Of these, 20 are encoded by the universal geneticcode (cf. table X above) and the remaining 2; selenocysteine (Sec, U)and pyrrolysine (Pyl, O), are incorporated into proteins by uniquesynthetic mechanisms.

A peptide according to the invention in one embodiment comprises one ormore non-naturally occurring amino acid residues (unnatural,non-proteinogenic or non-standard amino acids). Non-naturally occurringamino acids include e.g., without limitation, beta-2-naphthyl-alanine,trans-3-methylproline, 2,4-methanoproline, cis-4-hydroxyproline,ornithine, trans-4-hydroxyproline, N-methylglycine, allo-threonine,methylthreonine, hydroxyethylcysteine, hydroxyethylhomocysteine,nitroglutamnine, homoglutamine, pipecolic acid, thiazolidine carboxylicacid, dehydroproline, 3- and 4-methylproline, 3,3-dimethylproline,tert-leucine, norleucine, norvaline, 2-azaphenylalanine,3-azaphenylalanine, 4-azaphenylalanine, and 4-fluorophenylalanine.

Any amino acids according to the present invention may be in the L- orD-configuration. If nothing is specified, reference to the L-isomericform is preferably meant.

The standard and/or non-standard amino acids may be linked by peptidebonds (to form a linear peptide chain), or by non-peptide bonds (e.g.via the variable side-chains of the amino acids). Preferably, the aminoacids of the present invention are linked by peptide bonds.

The term peptide also embraces post-translational modificationsintroduced by chemical or enzyme-catalyzed reactions, as are known inthe art. These include acetylation, phosphorylation, methylation,glucosylation, glycation, amidation, hydroxylation, deimination,deamidation, carbamylation and sulfation of one or more amino acidresidues, and also proteolytic modification by known proteinasesincluding lysosomal kathepsins, and also calpains, secretases andmatrix-metalloproteinases.

In one embodiment the peptide of the present invention is amidated, suchas C-terminally amidated (—NH₂). In one exemplary embodiment thereof,the peptide is hGIP(3-30)—NH₂.

In one embodiment the peptide of the present invention is acetylated,such as N-terminally acetylated.

Also, functional equivalents of the peptides may comprise chemicalmodifications such as ubiquitination, labeling (e.g., withradionuclides, various enzymes, etc.), pegylation (derivatization withpolyethylene glycol), or by insertion (or substitution by chemicalsynthesis) of amino acids such as ornithine, which do not normally occurin human proteins (non-proteinogenic).

Sterically similar compounds may be formulated to mimic the key portionsof the peptide structure. This may be achieved by techniques ofmodelling and chemical designing known to those of skill in the art. Forexample, esterification and other alkylations may be employed to modifythe amino terminus of e.g. a di-arginine peptide backbone, to mimic atetra peptide structure. It will be understood that all such stericallysimilar constructs fall within the scope of the present invention.Peptides with N-terminal and C-terminal alkylations and esterificationsare also encompassed within the present invention.

A contiguous or consecutive peptide sequence is a sequence ofconsecutive amino acids being linked linearly by peptide bonds.Contiguous and consecutive amino acid sequence is used interchangeablyherein.

A peptide of the present invention in one embodiment consists of 21 to39 contiguous amino acids derived from GIP (SEQ ID NO: 4). In oneembodiment, the peptide according to the present invention comprises orconsists of a contiguous amino acid sequence of 39 amino acids, forexample 38 amino acids, such as 37 amino acids, for example 36 aminoacids, such as 35 amino acids, for example 34 amino acids, such as 33amino acids, for example 32 amino acids, such as 31 amino acids, forexample 30 amino acids, such as 29 amino acids, for example 28 aminoacids, such as 27 amino acids, for example 26 amino acids, such as 25amino acids, for example 24 amino acids, such as 23 amino acids, forexample 22 amino acids, such as 21 amino acids derived from GIP (SEQ IDNO:4) which comprises at least the sequence TFISDYSIAMDKIX₁QQDFVNW(GIP5-25, SEQ ID NO: 5) or a variant thereof.

In a particular embodiment the peptide of the invention consists of 28amino acids. In a particular embodiment the peptide of the inventionconsists of 28 amino acids corresponding to amino acids 3-30 of GIP (SEQID NO:4).

Compound of the Present Invention

It is an aspect of the present invention to provide a compoundcomprising or consisting of a peptide according to the presentinvention. In one embodiment, said peptide is formulated as a monomer(i.e. comprising 1 copy of the peptide), whereas in another embodiment,said peptide is formulated as a multimer.

Multimeric Compound

In one embodiment the peptide according to the present invention isformulated as a multimer. A multimer is a protein comprising orconsisting of multiple monomers. A multimer is an aggregate of multiplemolecules that is usually held together with non-covalent bonds. Thisdefinition distinguishes a multimer from a polymer, which is a series ofmonomers that are held together with covalent bonds.

A peptide sequence of the present invention is in one embodimentconnected to another (identical or non-identical) peptide sequence ofthe present invention by a chemical bond or through a linker group. Insome embodiments a peptide of the invention is formulated as an oligomeror multimer of monomers, wherein each monomer is as a peptide sequenceas defined according to the present invention.

Thus, according to the invention a multimeric compound is in oneembodiment a polymer comprising two or more peptide sequences of theinvention, said peptide sequences being identical or non-identical,wherein at least one of the two or more peptide sequences is a peptideaccording to the present invention. Preferably, both peptide sequencesare a peptide according to the present invention.

In one embodiment the multimeric compound is a dimer, comprising twopeptides according to the present invention, said two peptides beingidentical or non-identical with respect to each other.

In another embodiment the multimeric compound is a trimer, comprisingthree peptides according to the present invention, said peptides beingidentical or non-identical with respect to each other.

In another embodiment the multimeric compound is a tetramer, comprisingfour peptides according to the present invention, said peptides beingidentical or non-identical with respect to each other.

In one embodiment the multimeric compound is a dendrimer, such as atetrameric or octameric dendrimer. Dendrimers are repeatedly branched,roughly spherical large molecules, typically symmetric around the core,and often adopts a spherical three-dimensional morphology.

Dendrimers according to the present invention may comprise 4 peptides, 8peptides, 16 peptides, or 32 peptides. In one particular embodiment saiddendrimer comprises four peptides (i.e. a tetrameric dendrimer) or eightpeptides (octameric dendrimer).

In some particular embodiments, the multimeric compound comprises twoidentical amino acid sequences of the present invention (dimer) or thecompound comprises four identical copies of an amino acid sequence ofthe present invention (tetrameric dendrimer).

The multimers according to the invention is in one embodiment made bylinking two or more peptide monomers via a peptide bond or a linkergroup. In one embodiment they are linked to a lysine backbone, such as alysine residue (each peptide chain is linked to a single lysineresidue), or coupled to a polymer carrier, for example a proteincarrier. Said linker group in one embodiment comprises a plurality oflysine residues, such as a core moiety having a plurality of lysineresidues, such as seen in a lysine-based dendromeric structurecontaining three, seven, fifteen and more lysine residues However, anyother linking of peptide monomers known to the skilled person may beenvisioned.

The linking in one embodiment occurs at the N-terminal and/or C-terminalend of the peptide monomers.

Antagonist Activity of the Peptides

In some embodiments, the peptides according to the invention are capableof binding to and antagonizing a GIPR. The GIPR can be any GIPR,including the human GIPR (Uniprot accession number P48546), the mouseGIPR (Uniprot accession number Q0P543) and the rat GIPR (Uniprotaccession number P43219). In a preferred embodiment, the GIPR is thehuman GIPR (hGIPR).

Accordingly, the peptides of the invention will potentially reduce orprevent binding of full-length GIP1-42 and/or of GIP1-30 to the GIPR. Insome embodiments, the peptide potentially reduces or prevents binding offull-length hGIP1-42 (SEQ ID NO: 65) and/or of hGIP1-30 (SEQ ID NO: 69)to the hGIPR (or the rGIPR or mGIPR). In other embodiments, the peptidepotentially reduces or prevents binding of full-length rGIP1-42 (SEQ IDNO: 66) and/or of rGIP1-30 (SEQ ID NO: 70) to the rGIPR (or the hGIPR ormGIPR). In some embodiments, the peptide potentially reduces or preventsbinding of full-length mGIP1-42 (SEQ ID NO: 67) and/or of mGIP1-30 (SEQID NO: 71) to the mGIPR (or hGIPR or rGIPR).

The peptides of the invention in one embodiment are selected from thegroup consisting of competitive antagonists, uncompetitive antagonists,non-competitive antagonists, silent antagonists, partial agonists orinverse agonists. In a particular embodiment, the peptide is acompetitive antagonist of the GIPR.

Thus in some embodiments, the peptide is a competitive antagonist of thehGIPR. In other embodiments, the peptide is a competitive antagonist ofthe mGIPR. In other embodiments, the peptide is a competitive antagonistof the rGPIR. In other embodiments, the peptide is a competitiveantagonist of two or more of the hGIPR, the rGIPR and the rGPIR.

Antagonists have a Ki value which reflects the affinity of antagonistsfor their receptor and consequently their ability to inhibit agonistbinding. The peptide of the invention in one embodiment has a Ki of atleast 1 nM, such as at least 5 nM, 10 nM, such at least 15 nM, such asat least 20 nM, such as at least 25 nM, such as 30 nM, such as at least35 nM, such as 40 nM, such as at least 45 nM, such as at least 50 nM,such as at least 55 nM, such as at least 60 nM.

The peptide of the invention in one embodiment has a Ki of 1 to 200 nM,such as 1 to 5 nM, such as 5 to 10 nM, such as 10 to 15 nM, such as 15to 20 nM, such as 20 to 25 nM, such as 25 to 30 nM, such as 30 to 35 nM,such as 35 to 40 nM, such as 40 to 45 nM, such as 45 to 50 nM, such as50 to 55 nM, such as 55 to 60 nM, such as 60 to 65 nM, such as 65 to 70nM, such as 70 to 75 nM, such as 75 to 80 nM, such as 80 to 85, such as85 to 90 nM, such as 90 to 95 nM, such as 95 to 100 nM, such as 100 to105 nM, such as 105 to 110 nM, such as 110 to 115 nM, such as 115 to 120nM, such as 120 to 125 nM, such as 125 to 130 nM, such as 130 to 135 nM,such as 135 to 140 nM, such as 140 to 145 nM, such as 145 to 150 nM,such as 150 to 155 nM, such as 155 to 160 nM, such as 160 to 165 nM,such as 165 to 170 nM, such as 170 to 175 nM, such as 175 to 180 nM,such as 180 to 185 nM, such as 185 to 190 nM, such as 190 to 195 nM,such as 195 to 200 nM.

In some embodiments, the peptide has an affinity for a given GIPR whichis higher than the affinity of hGIP1-42 (SEQ ID NO: 65) for the sameGIPR. For example, the peptide in one embodiment has an affinity for thehGIPR, for the rGIPR and for the mGIPR which is higher than the affinityof hGIP1-42 (SEQ ID NO: 65) for the hGIPR, for the rGIPR and for themGIPR, respectively.

In some embodiments, the peptide has an affinity for a given GIPR whichis higher than the affinity of rGIP1-42 (SEQ ID NO: 59) for the sameGIPR. For example, the peptide in one embodiment has an affinity for thehGIPR, for the rGIPR and for the mGIPR which is higher than the affinityof rGIP1-42 (SEQ ID NO: 59) for the hGIPR, for the rGIPR and for themGIPR, respectively.

In some embodiments, the peptide has an affinity for a given GIPR whichis higher than the affinity of mGIP1-42 (SEQ ID NO: 60) for the sameGIPR. For example, the peptide in one embodiment has an affinity for thehGIPR, for the rGIPR and for the mGIPR which is higher than the affinityof mGIP1-42 (SEQ ID NO: 60) for the hGIPR, for the rGIPR and for themGIPR, respectively.

In some embodiments the peptide of the invention is capable ofdisplacing GIP1-42, GIP1-30 and/or GIP3-42 from a GIPR. In someembodiments the peptide of the invention is capable of displacing human,mouse or rat GIP1-42 and/or -GIP1-30 from the hGIPR, for the rGIPRand/or for the mGIPR. In one embodiment the peptide is capable ofdisplacing hGIP1-42 (SEQ ID NO: 65) from the hGIPR. In one embodimentthe peptide is capable of displacing rGIP1-42 (SEQ ID NO: 66) from therGIPR. In one embodiment the peptide is capable of displacing mGIP1-42(SEQ ID NO: 67) from the mGIPR.

In some embodiments, the peptide of the invention is capable ofdisplacing hGIP1-42 (SEQ ID NO: 65) and/or hGIP1-30 (SEQ ID NO: 69) withan IC50 value of at least 0.5 nM, for example approx. 0.92 nM, such asat least 1 nM, such as at least 2 nM, such as at least 3 nM, such as atleast 4 nM, such as at least 5 nM, such as at least 5.2 nM, such as atleast 6 nM, such as at least 7 nM, such as at least 7.8 nM, such as atleast 8 nM, such as at least 9 nM, such as at least 10 nM, such as atleast 11 nM, such as at least 11.4 nM, such as at least 12 nM, such asat least 13 nM, such as at least 14 nM, such as at least 15 nM, such asat least 16 nM.

In some embodiments, the peptide of the invention is capable ofdisplacing hGIP1-42 (SEQ ID NO: 65) and/or hGIP1-30 (SEQ ID NO: 69) withan IC50 value of 1 to 100 nM, such as 1 to 5 nM, such as 5 to 10 nM,such as 10 to 15 nM, such as 15 to 20 nM, such as 20 to 25 nM, such as25 to 30 nM, such as 30 to 35 nM, such as 35 to 40 nM, such as 40 to 45nM, such as 45 to 50 nM, such as 50 to 55 nM, such as 55 to 60 nM, suchas 60 to 65 nM, such as 65 to 70 nM, such as 70 to 75 nM, such as 75 to80 nM, such as 80 to 85 nM, such as 85 to 90 nM, such as 90 to 95 nM,such as 95 to 100 nM.

In some embodiments the peptide is capable of displacing hGIP1-42 (SEQID NO: 65) and/or hGIP1-30 (SEQ ID NO: 69) with an IC50 value of 1 to100 nM. In other embodiments the peptide is capable of displacingrGIP1-42 (SEQ ID NO: 66) and/or rGIP1-30 (SEQ ID NO: 70) with an IC50value of 1 to 100 nM. In other embodiments the peptide is capable ofdisplacing mGIP1-42 (SEQ ID NO: 67) and/or mGIP1-30 (SEQ ID NO: 71) withan IC50 value of 1 to 100 nM.

In some embodiments, the peptides of the invention are capable ofantagonizing somatostatin secretion induced by native GIP. In particularembodiments, the peptide is capable of antagonizing somatostatinsecretion induced by hGIP1-42 (SEQ ID NO: 65).

In some embodiments, the peptides of the invention are capable ofantagonizing insulin secretion induced by native GIP. In particularembodiments said peptide is capable of antagonising insulin secretioninduced by hGIP1-42 (SEQ ID NO: 65).

In some embodiments, the peptides of the invention are capable ofantagonizing glucagon secretion induced by native GIP. In particularembodiments said peptide is capable of antagonising glucagon secretioninduced by hGIP1-42 (SEQ ID NO: 65).

Without being bound by theory, it is believed that antagonizing the GIPRresults in reduced somatostatin levels and/or reduced insulin levelsand/or lower free fatty acid levels.

Determining Antagonist Properties and Affinity

In order to determine whether a peptide is an antagonist of the GIPR,methods known in the art may be employed, for example by determining theIC50 of the peptide. This can be done by constructing a dose-responsecurve and examining the effect of different concentrations of thepeptide on reversing agonist activity. The agonist can be GIP1-42, forexample hGIP-1-42 (SEQ ID NO: 65), rGIP1-42 (SEQ ID NO: 66) or mGIP1-42(SEQ ID NO: 67); or GIP1-30. The GIPR can be hGIPR, rGIPR or mGIPR. IC50values can be calculated for a given antagonist by determining theconcentration needed to inhibit half of the maximum biological responseof the agonist. A method for determining whether a peptide is anantagonist is described in example 4, but other methods known in the artmay also be used. For example, Schild plot analysis may be performed onhGIP1-42 (SEQ ID NO: 65) cAMP dose-response curves with increasingconcentrations of GIP-derived peptides. In this way, the type ofantagonist activity may also be determined.

Heterologous competition binding experiments may be performed in orderto measure the affinity of the peptide for a GIPR, i.e. how efficientlythe peptide is capable of displacing a given GIP1-42, for examplehGIP1-42 (SEQ ID NO: 65), rGIP1-42 (SEQ ID NO: 66) or mGIP1-42 (SEQ IDNO: 67). These competition binding experiments may be performed asdescribed in example 3 or by other methods known in the art. Forexample, GIP1-42 may be radioactively labelled, for example with ¹²⁵I.Other suitable isotopes are known to the skilled person.

Nucleic Acid Construct Encoding GIP Peptide

There are a variety of metabolic disorders and diseases arising fromgenetic and non-genetic causes, or a combination of both. Beta-cells ofthe pancreas are a possible target of gene therapy.

In one embodiment of the present invention there is provided a nucleicacid construct encoding a peptide according to the present invention.Preferably said nucleic acid construct will be able to continuouslyexpress a peptide according to the present invention for a prolongedperiod of time, such as at least 1 month, for example at least 2 months,such as at least 3 months, for example at least 4 months, such as atleast 5 months, for example at least 6 months, such as at least 7months, for example at least 8 months, such as at least 9 months, forexample at least 12 months.

It is an aspect of the present invention to provide a nucleic acidconstruct encoding a peptide according to the present invention.

In one embodiment there is provided a nucleic acid construct encoding apeptide selected from the group consisting of

(GIP3-30; SEQ ID NO: 74) EGTFISDYSIAMX _(0a) X _(0b)IX ₁QQDFVNWLLAQX ₂,(GIP4-30; SEQ ID NO: 75) GTFISDYSIAMX _(0a) X _(0b)IX ₁QQDFVNWLLAQX ₂,(GIP5-30; SEQ ID NO: 76) TFISDYSIAMX _(0a) X _(0b)IX ₁QQDFVNWLLAQX ₂,

wherein X_(0a), X_(0b), X₁ and X₂ are individually any amino acid,

or a functional variant thereof having at least 75% sequence identity tosaid peptide,

It is also an aspect of the present invention to provide a nucleic acidconstruct encoding a peptide according to the invention for use in amethod of treating metabolic syndrome such as obesity, diabetes, insulinresistance or fatty acid metabolism disorder, or of atherosclerosis, orof treating a cancer such as colon cancer or adrenal adenoma, or oftreating a bone density disorder such as a bone density disordercharacterized by high bone density and/or increased bone volume.

In one embodiment, the encoded peptide of the nucleic acid construct isa peptide according to the invention as defined herein elsewhere.

By nucleic acid construct is understood a genetically engineered nucleicacid. The nucleic acid construct may be a non-replicating and linearnucleic acid, a circular expression vector or an autonomouslyreplicating plasmid. A nucleic acid construct may comprise severalelements such as, but not limited to genes or fragments of same,promoters, enhancers, terminators, poly-A tails, linkers, polylinkers,operative linkers, multiple cloning sites (MCS), markers, STOP codons,internal ribosomal entry sites (IRES) and host homologous sequences forintegration or other defined elements. It is to be understood that thenucleic acid construct according to the present invention may compriseall or a subset of any combination of the above-mentioned elements.

Methods for engineering nucleic acid constructs are well known in theart (see, e.g., Molecular Cloning: A Laboratory Manual, Sambrook et al.,eds., Cold Spring Harbor Laboratory, 2nd Edition, Cold Spring Harbor,N.Y., 1989). Further, nucleic acid constructs according to the presentinvention may be synthesized without template, and may be obtained fromvarious commercial suppliers (e.g. Genscript Corporation).

In one embodiment, the nucleic acid construct are naked DNA constructscomprising sequences encoding the peptide of the invention.

Delivery Vehicles

It is also an aspect of the present invention to provide the nucleicacid construct as described herein above comprised within a deliveryvehicle. A delivery vehicle is an entity whereby a nucleotide sequenceor polypeptide or both can be transported from at least one media toanother. Delivery vehicles are generally used for expression of thesequences encoded within the nucleic acid construct and/or for theintracellular delivery of the construct or the polypeptide encodedtherein.

In one embodiment, there is provided a delivery vehicle comprising thenucleic acid construct according to the present invention. A deliveryvehicle may be selected from the group consisting of: RNA basedvehicles, DNA based vehicles/vectors, lipid based vehicles (such as aliposome), polymer based vehicles (such as a cationic polymer DNAcarrier), colloidal gold particles (coating) and virally derived DNA orRNA vehicles or vectors.

Methods of non-viral delivery include physical (carrier-free delivery)and chemical approaches (synthetic vector-based delivery).

Physical approaches, including needle injection, gene gun, jetinjection, electroporation, ultrasound, and hydrodynamic delivery,employ a physical force that permeates the cell membrane and facilitatesintracellular gene transfer. Said physical force may be electrical ormechanical.

Examples of chemical delivery vehicles include, but are not limited to:biodegradable polymer microspheres, lipid based formulations such asliposome carriers, cationically charged molecules such as liposomes,calcium salts or dendrimers, lipopolysaccharides, polypeptides andpolysaccharides.

Another embodiment of the present invention comprises a vector whichherein is denoted a viral vector (i.e. not a virus) as a deliveryvehicle. Viral vectors according to the present invention are made froma modified viral genome, i.e. the actual DNA or RNA forming the viralgenome, and introduced in naked form. Thus, any coat structuressurrounding the viral genome made from viral or non-viral proteins arenot part of the viral vector according to the present invention.

The virus from which the viral vector is derived may be selected fromthe non-exhaustive group of: adenoviruses, retroviruses, lentiviruses,adeno-associated viruses, herpesviruses, vaccinia viruses, foamyviruses, cytomegaloviruses, Semliki forest virus, poxviruses, RNA virusvector and DNA virus vector. Such viral vectors are well known in theart.

In one embodiment, said viral vectors may be selected from the groupconsisting of adenoviruses, lentiviruses, adeno-associated viruses (AAV)and recombinant adeno-associated viruses (rAAV). In one preferredembodiment, said viral vector is a therapeutic rAAV vector such as atherapeutic rAAV vector.

An adenovirus is a group of double-stranded DNA containing viruses.Adenoviruses can be genetically modified making them replicationincompetent or conditionally replication incompetent. In this form, asadenoviral constructs or adenovectors, they can be used as gene deliveryvehicles for vaccination or gene therapy.

Gene therapy vectors using AAV can infect both dividing and quiescentcells and persist in an extrachromosomal state without integrating intothe genome of the host cell. These features make AAV a very attractivecandidate for creating viral vectors for gene therapy. To date, AAVvectors have been used in over 80 clinical trials worldwide.

Recombinant Cell

Another aspect of the present invention relates to a cell comprising thenucleic acid construct according to the present invention. Such arecombinant cell can be used a tool for in vitro research, as a deliveryvehicle for the nucleic acid construct or as part of a gene-therapyregime. The nucleic acid construct according to the invention can beintroduced into cells by techniques well known in the art which includemicroinjection of DNA into the nucleus of a cell, transfection,electroporation, lipofection/liposome fusion and particle bombardment.Suitable cells include autologous and non-autologous cells, and mayinclude xenogenic cells.

Method of Treatment

It is an aspect of the present invention to provide a peptide, a nucleicacid construct encoding a peptide according to the present invention, adelivery vehicle comprising a nucleic acid construct encoding a peptideaccording to the present invention, as well a composition comprising thepeptide according to the invention, for use as a medicament.

It is a further aspect of the present invention to provide:

-   -   a. a peptide as defined herein,    -   b. a peptide consisting of 21 to 39 contiguous amino acid        residues derived from GIP (SEQ ID NO: 4), wherein said peptide        comprises at least the sequence TFISDYSIAMDKIX₁QQDFVNW (GIP5-25,        SEQ ID NO: 5), wherein X₁ is any amino acid, wherein said        peptide does not comprise the Tyr amino acid of position 1 of        SEQ ID NO: 4, and wherein said peptide does not comprise the Ala        amino acid of position 2 of SEQ ID NO: 4, or a functional        variant thereof having at least 60% identity to said peptide, or    -   c. a peptide selected from the group consisting of

(GIP3-30; SEQ ID NO: 74) EGTFISDYSIAMX _(0a) X _(0b)IX ₁QQDFVNWLLAQX ₂,(GIP4-30; SEQ ID NO: 75) GTFISDYSIAMX _(0a) X _(0b)IX ₁QQDFVNWLLAQX ₂,(GIP5-30; SEQ ID NO: 76) TFISDYSIAMX _(0a) X _(0b)IX ₁QQDFVNWLLAQX ₂,

-   -   wherein X_(0a), X_(0b), X₁ and X₂ are individually any amino        acid, or a functional variant thereof having at least 75%        sequence identity to said peptide,

for use in a method of inhibiting or reducing one or more of i)GIP-induced glucagon secretion, ii) GIP-induced insulin secretion, iii)GIP-induced somatostatin secretion, iv) GIP-induced glucose uptake, v)GIP-induced fatty acid synthesis and/or fatty acid incorporation, vi)high or increased expression or activity of a GIPR, vii) post-prandialGIP release, viii) serum levels of free fatty acids and/ortriglycerides, and ix) ix) GIP-induced reduction in bone resorption.

It is a further aspect of the present invention to provide:

-   -   a. a peptide as defined herein,    -   b. a peptide consisting of 21 to 39 contiguous amino acid        residues derived from GIP (SEQ ID NO: 4), wherein said peptide        comprises at least the sequence TFISDYSIAMDKIX₁QQDFVNW (GIP5-25,        SEQ ID NO: 5), wherein X₁ is any amino acid, wherein said        peptide does not comprise the Tyr amino acid of position 1 of        SEQ ID NO: 4, and wherein said peptide does not comprise the Ala        amino acid of position 2 of SEQ ID NO: 4, or a functional        variant thereof having at least 60% identity to said peptide, or    -   c. a peptide selected from the group consisting of

(GIP3-30; SEQ ID NO: 74) EGTFISDYSIAMX _(0a) X _(0b)IX ₁QQDFVNWLLAQX ₂,(GIP4-30; SEQ ID NO: 75) GTFISDYSIAMX _(0a) X _(0b)IX ₁QQDFVNWLLAQX ₂,(GIP5-30; SEQ ID NO: 76) TFISDYSIAMX _(0a) X _(0b)IX ₁QQDFVNWLLAQX ₂,

-   -   wherein X_(0a), X_(0b), X₁ and X₂ are individually any amino        acid, or a functional variant thereof having at least 75%        sequence identity to said peptide,

for use in a method of treating metabolic syndrome.

In one embodiment the metabolic syndrome is selected from the groupconsisting of obesity, obesity-related disorders, pre-diabetes (impairedfasting glucose), diabetes mellitus, diabetes-related disorders, insulinresistance, elevated fasting glucose (hyperglycemia), elevated fastingserum triglyceride level (VLDL triglyceride), low high-densitylipoprotein (HDL) levels, fatty acid metabolism disorder, cardiovasculardisease, elevated blood pressure and atherosclerosis.

In one embodiment the metabolic syndrome is obesity.

In one embodiment the metabolic syndrome is diabetes mellitus, includingdiabetes mellitus type I and type II.

In one embodiment the metabolic syndrome is insulin resistance.

In one embodiment the metabolic syndrome is a fatty acid metabolismdisorder.

It is a further aspect of the present invention to provide a peptide asdefined herein for use in a method of treating cancer.

In one embodiment the cancer is selected from the group consisting ofcolon cancer, a neuroendocrine cancer and adrenal adenoma.

It is a further aspect of the present invention to provide a peptide asdefined herein for use in a method of treating a bone density disorder.

In one embodiment there is provided a peptide as defined herein for usein a method of inhibiting activity of bone cells. In one embodimentthere is provided a peptide as defined herein for use in a method ofinhibiting (or antagonizing) GIP-induced postprandial reduction in boneresorption. In one embodiment there is provided a peptide as definedherein for use in a method of treating bone cancer.

In one embodiment, the bone density disorder is selected from the groupconsisting of atherosclerosis, disorders characterized by low bonedensity and/or reduced bone volume, disorders characterized by high bonedensity and/or increased bone volume and osteoporosis.

In yet another aspect, the invention relates to the use of such peptidesin a method of characterizing or examining aspects of a disorder, and/orcharacterizing or examining aspects of the human physiology associatedwith a disorder, wherein said disorder in one embodiment is selectedfrom metabolic disorder or syndrome, such as obesity, diabetes mellitus,insulin resistance or fatty acid metabolism disorder. In other aspectsthe invention relates to methods of treating cancer, such as coloncancer or adrenal adenoma. In other aspects the invention relates tomethods of treating a bone density disorder characterized by high bonedensity and/or increased bone volume or osteoporosis. In other aspectsthe invention relates to methods of treating atherosclerosis.

It is another aspect to provide a peptide according to the invention forthe manufacture of a medicament.

In one embodiment there is provided the use of a peptide according tothe invention in the manufacture of a medicament for inhibiting orreducing one or more of i) GIP-induced glucagon secretion, ii)GIP-induced insulin secretion, iii) GIP-induced somatostatin secretion,iv) GIP-induced glucose uptake, v) GIP-induced fatty acid synthesisand/or fatty acid incorporation, vi) high or increased expression oractivity of a GIPR, vii) post-prandial GIP release, viii) serum levelsof free fatty acids and/or triglycerides and ix) ix) GIP-inducedreduction of bone resorption.

Also provided is a method for treating metabolic syndrome such asobesity, diabetes mellitus, insulin resistance or fatty acid metabolismdisorder; a cancer such as colon cancer or adrenal adenoma; a bonedensity disorder, such as bone density disorders characterized by highbone density and/or increased bone volume; or atherosclerosis; saidmethod comprising the step of administering to an individual in needthereof an effective amount of a peptide as defined herein.

An individual in need as referred to herein, is an individual that maybenefit from the administration of a peptide or pharmaceuticalcomposition according to the present invention. Such an individual maysuffer from a metabolic disorder such as obesity, diabetes, insulinresistance or fatty acid metabolism disorder, a cancer such as coloncancer or adrenal adenoma, a bone density disorder, or be in risk ofsuffering therefrom. The individual may be any human being, male orfemale, infant, middle-aged or old. The disorder to be treated orprevented in the individual may relate to the age of the individual, thegeneral health of the individual, the medications used for treating theindividual and whether or not the individual has a prior history ofsuffering from diseases or disorders that may have or have induced ametabolic disorder such as obesity, diabetes, insulin resistance orfatty acid metabolism disorder, a cancer such as colon cancer or adrenaladenoma, atherosclerosis, a bone density disorder. In some embodiments,the disorder to be treated is linked to GIP-induced glucagon secretion,GIP-induced insulin secretion, to GIP-induced somatostatin secretion, toGIP-induced glucose uptake, to GIP-induced fatty acid synthesis and/orfatty acid incorporation, to high expression and/or activity of a GIPR,to release of GIP following a meal; wherein the term “high” is to beconstrued as referring to levels greater than the corresponding levelsobserved in individuals not in need of treatment.

Method of Preparation (Peptide)

The peptides according to the present invention may be prepared by anymethods known in the art. Thus, the GIP-derived peptides may be preparedby standard peptide-preparation techniques such as solution synthesis orMerrifield-type solid phase synthesis.

In one embodiment, a peptide according to the invention is anon-naturally occurring peptide; being derived from a naturallyoccurring protein native GIP.

In another embodiment, the peptide according to the invention is anaturally occurring peptide being derived from a naturally occurringprotein GIP1-42.

In one embodiment a peptide according to the present invention ispurified from a naturally occurring source thereof, such as serum.Protein purification is a series of processes intended to isolate asingle type of protein from a complex mixture. The starting material isusually a biological tissue. The various steps in the purificationprocess may free the protein from a matrix that confines it, separatethe protein and non-protein parts of the mixture, and finally separatethe desired protein from all other proteins. Separation steps mayexploit differences in (for example) protein size, physico-chemicalproperties, binding affinity and biological activity.

In one embodiment a peptide according to the invention is syntheticallymade or produced.

The methods for synthetic production of peptides are well known in theart. Detailed descriptions as well as practical advice for producingsynthetic peptides may be found in Synthetic Peptides: A User's Guide(Advances in Molecular Biology), Grant G. A. ed., Oxford UniversityPress, 2002, or in: Pharmaceutical Formulation: Development of Peptidesand Proteins, Frokjaer and Hovgaard eds., Taylor and Francis, 1999.

In one embodiment the peptide or peptide sequences of the invention areproduced synthetically, in particular, by the Sequence Assisted PeptideSynthesis (SAPS) method, by solution synthesis, by Solid-phase peptidesynthesis (SPPS) such as Merrifield-type solid phase synthesis, byrecombinant techniques (production by host cells comprising a firstnucleic acid sequence encoding the peptide operably associated with asecond nucleic acid capable of directing expression in said host cells)or enzymatic synthesis. These are well-known to the skilled person.

Peptides may be synthesised either batch-wise on a fully automatedpeptide synthesiser using 9-fluorenylmethyloxycarbonyl (Fmoc) ortert-Butyloxycarbonyl (Boc) as N-a-amino protecting group and suitablecommon protection groups for side-chain functionalities.

After purification such as by reversed phase HPLC, peptides may befurther processed to obtain for example cyclic or C- or N-terminalmodified isoforms. The methods for cyclization and terminal modificationare well-known in the art.

Peptides according to the invention may be synthesized as monomers ormultimers such as dimers or tetramers.

Pharmaceutical Composition and Formulation

Whilst it is possible for the bioactive agent of the present invention(a peptide, a nucleic acid construct encoding said peptide, and acomposition comprising a peptide) to be administered as the raw chemical(peptide), it is sometimes preferred to present them in the form of apharmaceutical formulation. Such a pharmaceutical formulation may bereferred to as a pharmaceutical composition, pharmaceutically acceptablecomposition or pharmaceutically safe composition.

Accordingly, the present invention further provides a pharmaceuticalformulation, which comprises a bioactive agent of the present invention,or a pharmaceutically acceptable salt or ester thereof, and apharmaceutically acceptable carrier, excipient and/or diluent. Thepharmaceutical formulations may be prepared by conventional techniques,e.g. as described in Remington: The Science and Practice of Pharmacy2005, Lippincott, Williams & Wilkins.

Pharmaceutically acceptable salts of the instant peptide compounds,where they can be prepared, are also intended to be covered by thisinvention. These salts will be ones which are acceptable in theirapplication to a pharmaceutical use. By that it is meant that the saltwill retain the biological activity of the parent compound and the saltwill not have untoward or deleterious effects in its application and usein treating diseases.

Pharmaceutically acceptable salts are prepared in a standard manner. Ifthe parent compound is a base it is treated with an excess of an organicor inorganic acid in a suitable solvent. If the parent compound is anacid, it is treated with an inorganic or organic base in a suitablesolvent.

The peptide compounds of the invention may be administered in the formof an alkali metal or earth alkali metal salt thereof, concurrently,simultaneously, or together with a pharmaceutically acceptable carrieror diluent, especially and preferably in the form of a pharmaceuticalcomposition thereof, whether by oral, rectal, or parenteral (includingsubcutaneous) route, in an effective amount.

Examples of pharmaceutically acceptable acid addition salts for use inthe present inventive pharmaceutical composition include those derivedfrom mineral acids, such as hydrochloric, hydrobromic, phosphoric,metaphosphoric, nitric and sulfuric acids, and organic acids, such astartaric, acetic, citric, malic, lactic, fumaric, benzoic, glycolic,gluconic, succinic, p-toluenesulphonic acids, and arylsulphonic, forexample.

In a preferred embodiment, the peptide according to the invention isformulated as an acetate salt.

The pharmaceutically acceptable salt of the peptide of the invention ispreferably in solution with a physiologically acceptable pH, i.e. thesolution comprising the peptide salt preferably has a pH acceptable forclinical use. For example, the salt may be diluted in 1 mM HCl and 0.1%human serum albumin (HSA) at pH 3.4 to a final concentration of 1.9mg/mL. This solution may be further diluted with 0.2% HSA saline to0.0162 mg/mL and the resulting solution preferably has physiologicallyacceptable pH. The final concentration of the peptide is preferably 0.2mM.

In one embodiment, the peptide composition according to the inventionmay be diluted in a solution with a final concentration of peptide atleast 0.05 mM, such as at least 0.1 mM, such as at least 0.2 mM, such asat least 0.3 mM, such as at least 0.4 mM, such as at least 0.5 mM. In apreferred embodiment, the final concentration of the peptide is 0.2 mM.

In another embodiment, the peptide composition according to theinvention may be diluted in a solution with a final concentration ofpeptide of 0.05 to 10 mM, such as 0.05 to 0.1 mM, such as 0.1 to 0.2 mM,such as 0.2 to 0.3 mM, such as 0.3 to 0.4 mM, such as 0.4 to 0.5 mM,such as 0.5 to 0.6 mM, such as 0.6 to 0.7 mM, such as 0.7 to 0.8 mM,such as 0.8 to 0.9 mM, such as 0.9 to 1 mM, such as 1 to 2 mM, such as 2to 3 mM, such as 3 to 4 mM, such as 4 to 5 mM, such as 5 to 6 mM, suchas 6 to 7 mM, such as 7 to 8 mM, such as 8 to 9 mM, such as 9 to 10 mM.

In a preferred embodiment, the peptide is diluted in HSA saline at afinal concentration of 0.2 mM, the resulting solution having aphysiologically acceptable pH such as pH 6.7.

In one embodiment the pharmaceutical formulation of the presentinvention has a pH in the range of 5.5 to 8, such as 5.5 to 6, such as 6to 6.5, for example 6.5 to 7, such as 7 to 7.5, for example 7.5 to 8.

Administration and Dosage

According to the present invention, a peptide or a nucleic acidconstruct encoding said peptide, or a composition comprising a peptideas defined herein is administered to individuals in need of treatment inpharmaceutically effective doses or a therapeutically effective amount.The dosage requirements will vary with the particular drug compositionemployed, the route of administration and the particular subject beingtreated, which depend on the severity and the sort of the disorder aswell as on the weight and general state of the subject. It will also berecognized by one skilled in the art that the optimal quantity andspacing of individual dosages of a peptide compound will be determinedby the nature and extent of the condition being treated, the form, routeand site of administration, and the particular patient being treated,and that such optima can be determined by conventional techniques. Itwill also be appreciated by one of skill in the art that the optimalcourse of treatment, i.e., the number of doses of a compound given perday for a defined number of days, can be ascertained using conventionalcourse of treatment determination tests.

In one embodiment of the present invention, the bioactive agent isadministered at least once daily, such as once daily, such as twicedaily, such as thrice daily, such as four times daily, such as fivetimes daily.

A dose may also be administered in intermittent intervals, or intervals,whereby a dose is not administered every day. Rather one or more dosesmay be administered every second day, every third day, every fourth day,every fifth day, every sixth day, every week, every second week, everythird week, every fourth week, every fifth week, every sixth week, orintervals within those ranges (such as every 2 to 4 weeks, or 4 to 6weeks).

In one embodiment of the present invention, the bioactive agent isadministered in doses of at least 30000 pmol/kg/day, such as at least60000 pmol/kg/day, such as at least 72000 pmol/kg/day, such as at least90000 pmol/kg/day, such as at least 120000 pmol/kg/day, such as at least150000 pmol/kg/day, such as at least 30000 pmol/kg/day, preferably suchas at least 60000 pmol/kg/day. In a particular embodiment, the bioactiveagent is administered at a dosage of 72000 pmol/kg/day.

In one embodiment, the bioactive agent is administered at a daily dosageof 30000 pmol/kg to 40000 pmol/kg, such as 40000 pmol/kg to 50000pmol/kg, such as 50000 pmol/kg to 60000 pmol/kg, such as 60000 pmol/kgto 70000 pmol/kg, such as 70000 pmol/kg to 80000 pmol/kg, such as 80000pmol/kg to 90000 pmol/kg, such as 90000 pmol/kg to 100000 pmol/kg, suchas 100000 pmol/kg to 110000 pmol/kg, such as 110000 pmol/kg to 120000pmol/kg. In a particular embodiment, the bioactive agent is a peptideand is administered at a daily dose of 60000 pmol/kg or 72000 pmol/kg.

In one embodiment of the present invention, the bioactive agent isadministered by infusion. In one embodiment, the bioactive agent is apeptide, and the infusion takes place over a duration of at least 15min, such as at least 20 min, such as at least 30 min, such as at least40 min, such as at least 50 min, such as at least 60 min, such as atleast 90 min, such as at least 120 min, preferably such as 60 min.

In one embodiment of the present invention, the bioactive agent isadministered over a duration between 15 and 120 min, such as between 15and 20 min, such as between 20 and 30 min, such as between 30 and 40min, such as between 40 and 50 min, such as between 50 and 60 min, suchas between 60 and 90 min, such as between 90 and 120 min.

In one embodiment, the bioactive agent is administered once daily over aduration of 60 min, or twice daily over a duration of 30 min, or thricedaily over a duration of 20 min, or four times daily over a duration of15 min, or five times daily over a duration of 12 min, where theduration is the duration of each individual administration.

In one embodiment, the bioactive agent is administered at a dosage of atleast 500 pmol/kg/min, such as at least 1000 pmol/kg/min, such as atleast 1200 pmol/kg/min, such as at least 1500 pmol/kg/min, such as atleast 2000 pmol/kg/min, such as at least 2500 pmol/kg/min, such as atleast 5000 pmol/kg/min.

The skilled person knows that if the number of daily administrations isincreased, the dose to be administered in each administration may bedecreased accordingly. Likewise, if the duration of each administrationis decreased, the dosage may be increased accordingly.

The bioactive agent to be administered is a peptide according to thepresent invention.

In preferred embodiments, the peptide is hGIP3-30 (SEQ ID NO: 1),rGIP3-30 (SEQ ID NO: 2) or mGIP3-30 (SEQ ID NO: 3), or a functionalvariant thereof.

In one embodiment the bioactive agent is administered with one or moreadditional active ingredients. These other ingredients may bepharmaceutically active. In some embodiments, the bioactive agent is apeptide as defined above and the other ingredient is hGIP1-42 (SEQ IDNO: 66) or a variant thereof. In some embodiments, hGIP1-42 isadministered at a dosage of at least 120 pmol/kg/day, such as at least130 pmol/kg/day, such as at least 140 pmol/kg/day, such as at least 150pmol/kg/day, such as at least 160 pmol/kg/day, such as at least 170pmol/kg/day, such as at least 180 pmol/kg/day, such as at least 190pmol/kg/day, such as at least 200 pmol/kg/day. In a preferredembodiment, hGIP1-42 is administered at a dosage of 120 pmol/kg/day. Inanother preferred embodiment, the bioactive agent is hGIP3-30 (SEQ IDNO: 1), rGIP3-30 (SEQ ID NO: 2) or mGIP3-30 (SEQ ID NO: 3), or afunctional variant thereof and the other ingredient is hGIP1-42 (SEQ IDNO: 65). In a preferred embodiment, the bioactive agent is hGIP3-30 (SEQID NO: 1), or a functional variant thereof and the other ingredient ishGIP1-42 (SEQ ID NO: 65) administered at a dosage of at least 120pmol/kg/day.

Routes of Administration

It will be appreciated that the preferred route of administration willdepend on the general condition and age of the subject to be treated,the nature of the condition to be treated, the location of the tissue tobe treated in the body and the active ingredient chosen.

Systemic Treatment

For systemic treatment according to the present invention the route ofadministration is capable of introducing the bioactive agent (a peptide,a nucleic acid construct encoding said peptide, and a compositioncomprising a peptide according to the present invention) into the bloodstream to ultimately target the sites of desired action.

Such routes of administration are any suitable routes, such as anenteral route (including the oral, rectal, nasal, pulmonary, buccal,sublingual, transdermal, intracisternal and intraperitonealadministration), and/or a parenteral route (including subcutaneous,intramuscular, intrathecal, intracerebral, intravenous and intradermaladministration).

Parenteral Administration

Parenteral administration is any administration route not being theoral/enteral route whereby the medicament avoids first-pass degradationin the liver. Accordingly, parenteral administration includes anyinjections and infusions, for example bolus injection or continuousinfusion, such as intravenous administration, intramuscularadministration or subcutaneous administration. Furthermore, parenteraladministration includes inhalations and topical administration.

Accordingly, the bioactive agent may be administered topically to crossany mucosal membrane of an animal to which the biologically activesubstance is to be given, e.g. in the nose, vagina, eye, mouth, genitaltract, lungs, gastrointestinal tract, or rectum, preferably the mucosaof the nose, or mouth, and accordingly, parenteral administration mayalso include buccal, sublingual, nasal, rectal, vaginal andintraperitoneal administration as well as pulmonal and bronchialadministration by inhalation or installation. Also, the agent may beadministered topically to cross the skin.

Local Treatment

The bioactive agent according to the invention may in one embodiment beused as a local treatment, i.e. be introduced directly to the site(s) ofaction. Accordingly, the bioactive agent may be applied to the skin ormucosa directly, or the bioactive agent may be injected into the site ofaction, for example into the diseased tissue or to an end artery leadingdirectly to the diseased tissue.

These administration forms preferably avoid the blood brain barrier.

Kit-of-Parts

The present invention also relates to a kit-of-parts comprising one ormore of the bioactive agents described above (a peptide, a nucleic acidconstruct or a composition), and at least one additional or furthercomponent.

A kit of parts according to the present invention comprises one or moreof the bioactive agents as defined herein for treatment, prevention oralleviation of a metabolic disorder such as obesity or diabetesmellitus, a bone density disorder or cancer. Kits according to thepresent invention allows for simultaneous, sequential or separateadministration of the bioactive agent according to the present inventionand/or one or more second active ingredients as described hereinelsewhere.

Items

-   -   1. A peptide consisting of 21 to 39 contiguous amino acid        residues derived from gastric inhibitory peptide (GIP) (SEQ ID        NO: 4), wherein said peptide comprises at least the sequence

(GIP5-25, SEQ ID NO: 5) TFISDYSIAMDKIX ₁QQDFVNW,

-   -   -   wherein X₁ is any amino acid,        -   wherein said peptide does not comprise the Tyr amino acid of            position 1 of SEQ ID NO: 4, and wherein said peptide does            not comprise the Ala amino acid of position 2 of SEQ ID NO:            4,        -   or a functional variant thereof having at least 60% identity            to said peptide.

    -   2. The peptide according to claim 1, wherein said peptide is        non-naturally occurring.

    -   3. The peptide according to any one of the preceding items,        wherein said peptide is synthetic.

    -   4. The peptide according to any one of the preceding items,        wherein said peptide is selected from the group consisting of:

(GIP3-25, SEQ ID NO: 6) EGTFISDYSIAMDKIX ₁QQDFVNW,(GIP3-26, SEQ ID NO: 7) EGTFISDYSIAMDKIX ₁QQDFVNWL,(GIP3-27, SEQ ID NO: 8) EGTFISDYSIAMDKIX ₁QQDFVNWLL,(GIP3-28, SEQ ID NO: 9) EGTFISDYSIAMDKIX ₁QQDFVNWLLA,(GIP3-29, SEQ ID NO: 10) EGTFISDYSIAMDKIX ₁QQDFVNWLLAQ,(GIP3-30, SEQ ID NO: 11) EGTFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂,(GIP3-31, SEQ ID NO: 12) EGTFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂G,(GIP3-32, SEQ ID NO: 13) EGTFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂GK,(GIP3-33, SEQ ID NO: 14) EGTFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂GKK,(GIP3-34, SEQ ID NO: 15) EGTFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂GKKN,(GIP3-35, SEQ ID NO: 16) EGTFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂GKKND,(GIP3-36, SEQ ID NO: 17) EGTFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂GKKNDW,(GIP3-37, SEQ ID NO: 18) EGTFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂GKKNDWK,(GIP3-38, SEQ ID NO: 19) EGTFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂GKKNDWKH,(GIP3-39, SEQ ID NO: 20) EGTFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂GKKNDWKHN,(GIP3-40, SEQ ID NO: 21) EGTFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂GKKNDWKHNI,(GIP3-41, SEQ ID NO: 22) EGTFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂GKKNDWKHNIT,

-   -   -   or a functional variant thereof, wherein X₁ and X₂ are            individually any amino acid.

    -   5. The peptide according to any one of the preceding items,        wherein said peptide is EGTFISDYSIAMDKIX₁QQDFVNWLLAQX₂ (GIP3-30,        SEQ ID NO: 11), or variants thereof.

    -   6. The peptide according to any one of the preceding items,        wherein said peptide is selected from the group consisting of:

(GIP4-25, SEQ ID NO: 23) GTFISDYSIAMDKIX ₁QQDFVNW,(GIP4-26, SEQ ID NO: 24) GTFISDYSIAMDKIX ₁QQDFVNWL,(GIP4-27, SEQ ID NO: 25) GTFISDYSIAMDKIX ₁QQDFVNWLL,(GIP4-28, SEQ ID NO: 26) GTFISDYSIAMDKIX ₁QQDFVNWLLA,(GIP4-29, SEQ ID NO: 27) GTFISDYSIAMDKIX ₁QQDFVNWLLAQ,(GIP4-30, SEQ ID NO: 28) GTFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂,(GIP4-31, SEQ ID NO: 29) GTFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂G,(GIP4-32, SEQ ID NO: 30) GTFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂GK,(GIP4-33, SEQ ID NO: 31) GTFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂GKK,(GIP4-34, SEQ ID NO: 32) GTFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂GKKN,(GIP4-35, SEQ ID NO: 33) GTFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂GKKND,(GIP4-36, SEQ ID NO: 34) GTFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂GKKNDW,(GIP4-37, SEQ ID NO: 35) GTFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂GKKNDWK,(GIP4-38, SEQ ID NO: 36) GTFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂GKKNDWKH,(GIP4-39, SEQ ID NO: 37) GTFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂GKKNDWKHN,(GIP4-40, SEQ ID NO: 38) GTFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂GKKNDWKHNI,(GIP4-41, SEQ ID NO: 39) GTFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂GKKNDWKHNIT,(GIP4-42, SEQ ID NO: 40) GTFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂GKKNDWKHNITQ,

-   -   -   or a functional variant thereof, wherein X₁ and X₂ are            individually any amino acid.

    -   7. The peptide according to any one of the preceding items,        wherein said peptide is selected from the group consisting of:

(GIP5-25, SEQ ID NO: 5) TFISDYSIAMDKIX ₁QQDFVNW,(GIP5-26, SEQ ID NO: 41) TFISDYSIAMDKIX ₁QQDFVNWL,(GIP5-27, SEQ ID NO: 42) TFISDYSIAMDKIX ₁QQDFVNWLL,(GIP5-28, SEQ ID NO: 43) TFISDYSIAMDKIX ₁QQDFVNWLLA,(GIP5-29, SEQ ID NO: 44) TFISDYSIAMDKIX ₁QQDFVNWLLAQ,(GIP5-30, SEQ ID NO: 45) TFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂,(GIP5-31, SEQ ID NO: 46) TFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂G,(GIP5-32, SEQ ID NO: 47) TFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂GK,(GIP5-33, SEQ ID NO: 48) TFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂GKK,(GIP5-34, SEQ ID NO: 49) TFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂GKKN,(GIP5-35, SEQ ID NO: 50) TFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂GKKND,(GIP5-36, SEQ ID NO: 51) TFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂GKKNDW,(GIP5-37, SEQ ID NO: 52) TFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂GKKNDWK,(GIP5-38, SEQ ID NO: 53) TFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂GKKNDWKH,(GIP5-39, SEQ ID NO: 54) TFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂GKKNDWKHN,(GIP5-40, SEQ ID NO: 55) TFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂GKKNDWKHNI,(GIP5-41, SEQ ID NO: 56) TFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂GKKNDWKHNIT,(GIP5-42, SEQ ID NO: 57) TFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂GKKNDWKHNITQ,

-   -   -   or a functional variant thereof, wherein X₁ and X₂ are            individually any amino acid.

    -   8. The peptide according to any one of the preceding items,        wherein X₁ is selected from the group consisting of Ala, His,        Arg and Lys.

    -   9. The peptide according to any one of the preceding items,        wherein X₂ is selected from the group consisting of Ala, Lys and        Arg.

    -   10. The peptide according to any one of the preceding items,        wherein X₁ is selected from the group consisting of His and Arg        and X₂ is Arg.

    -   11. The peptide according to any one of the preceding items,        wherein X₁ is His and X₂ is Lys.

    -   12. The peptide according to any one of the preceding items,        wherein X₁ is Arg and X₂ is Lys or Arg.

    -   13. The peptide according to any one of the preceding items,        wherein said peptide is selected from the group consisting of        EGTFISDYSIAMDKIHQQDFVNWLLAQK (hGIP3-30, SEQ ID NO: 1),        EGTFISDYSIAMDKIRQQDFVNWLLAQK (rGIP3-30, SEQ ID NO: 2),        EGTFISDYSIAMDKIRQQDFVNWLLAQR (mGIP3-30, SEQ ID NO: 3), and        variants thereof.

    -   14. The peptide according to any one of the preceding items,        wherein said peptide is hGIP3-30 (SEQ ID NO: 1) or a variant        thereof.

    -   15. The peptide according to any one of the preceding items,        wherein said peptide is rGIP3-30/hGIP(3-30)H18R (SEQ ID NO: 2)        or a variant thereof.

    -   16. The peptide according to any one of the preceding items,        wherein said peptide is mGIP3-30/hGIP(3-30)H18R/K30R (SEQ ID        NO: 3) or a variant thereof.

    -   17. The peptide according to any one of the preceding items,        wherein said peptide is amidated.

    -   18. The peptide according to any one of the preceding items,        wherein said peptide has at least 60% identity, such as at least        65% identity, such as at least 70% identity, such as at least        75% identity, such as at least 80% identity, such as at least        85% identity, such as at least 90% identity, such as at least        95% identity, such as at least 99% identity, such as 100%        identity to hGIP3-30 (SEQ ID NO: 1) and/or the corresponding        part of hGIP (SEQ ID NO:65).

    -   19. The peptide according to any one of the preceding items,        wherein said peptide comprises the sequence        TFISDYSIAMX_(0a)X_(0b)IX₁QQDFVNW, wherein X_(0a) and/or X_(0b)        are individually any amino acid.

    -   20. The peptide according to any one of the preceding items,        wherein D at position 15 (X_(0a)) and/or K at position 16        (X_(0b)) is substituted with a different amino acid, such as A        (Ala).

    -   21. The peptide according to any one of the preceding items,        wherein said peptide consists of 21 to 22 amino acids, for        example 22 to 23, such as 23 to 24, for example 24 to 25, such        as 25 to 26, for example 26 to 27, such as 27 to 28, for example        28 to 29, such as 29 to 30, for example 30 to 31, such as 31 to        32, for example 32 to 33, such as 33 to 34, for example 34 to        35, such as 35 to 36, for example 36 to 37, such as 37 to 38,        for example 38 to 39 contiguous amino acids.

    -   22. The peptide according to any one of the preceding items,        wherein said peptide binds to and/or is an antagonist of one or        more GIP-receptors (GIPRs), such as one or more of the human        GIPR (Uniprot accession number P48546), the rat GIPR (Uniprot        accession number P43219) and the murine GIPR (Uniprot accession        number Q0P543).

    -   23. The peptide according to any one of the preceding items,        wherein said peptide is a competitive antagonist, an        uncompetitive antagonist, a non-competitive antagonist, a silent        antagonist, a partial agonist or an inverse agonist of one or        more GIP-receptors (GIPRs), such as one or more of the human        GIPR (Uniprot accession number P48546), the rat GIPR (Uniprot        accession number P43219) and the murine GIPR (Uniprot accession        number Q0P543).

    -   24. The peptide according to item 23, wherein said peptide is a        competitive antagonist of one or more GIP-receptors (GIPRs).

    -   25. The peptide according to any one of the preceding items,        wherein said peptide has a Ki of at least 1 nM, such as at least        5 nM, such as at least 10 nM, such at least 15 nM, such as 20        nM, such as at least 25 nM, such as 30 nM, such as at least 35        nM, such as 40 nM, such as at least 45 nM, such as at least 50        nM, such as at least 55 nM.

    -   26. The peptide according to any one of the preceding items,        wherein said peptide has an affinity for a GIPR which is higher        than the affinity of GIP3-42 (SEQ ID NO: 58) for the same GIPR,        such as wherein said peptide has an affinity for the hGIPR which        is higher than the affinity of hGIP3-42 (SEQ ID NO: 62) for the        hGIPR.

    -   27. The peptide according to any one of the preceding items,        wherein said peptide is capable of displacing hGIP1-42 (SEQ ID        NO: 65) from the hGIPR, such as displacing hGIP1-42 with an IC50        value of at least 0.5 nM, such as at least 1 nM, such as at        least 2 nM, such as at least 3 nM, such as at least 4 nM, such        as at least 5 nM, such as at least 5.2 nM, such as at least 6        nM, such as at least 7 nM, such as at least 7.8 nM, such as at        least 8 nM, such as at least 9 nM, such as at least 10 nM, such        as at least 11 nM, such as at least 11.4 nM, such as at least 12        nM, such as at least 13 nM, such as at least 14 nM, such as at        least 15 nM, such as at least 16 nM.

    -   28. The peptide according to any one of the preceding items,        wherein said peptide is capable of inhibiting and/or        antagonising somatostatin secretion induced by hGIP1-42 (SEQ ID        NO: 65).

    -   29. The peptide according to any one of the preceding items,        wherein said peptide is capable of inhibiting and/or        antagonising insulin secretion induced by hGIP1-42 (SEQ ID NO:        65).

    -   30. The peptide according to any one of the preceding items,        wherein said peptide is capable of inhibiting and/or        antagonising glucagon secretion induced by hGIP1-42 (SEQ ID NO:        65).

    -   31. A nucleic acid construct encoding a peptide according to any        one of items 1 to 31.

    -   32. A delivery vehicle comprising the nucleic acid construct        according to item 32.

    -   33. A cell comprising the nucleic acid construct according to        item 32.

    -   34. A pharmaceutically acceptable composition comprising a        peptide according to any one of items 1 to 31.

    -   35. The composition according to item 35, said composition        comprising said peptide formulated as an acetate salt.

    -   36. The composition according to any one of items 35 to 38,        wherein said peptide is diluted in human serum albumin saline.

    -   37. The composition according to any one of items 35 to 39,        wherein the pH of the composition is acceptable for clinical        use.

    -   38. The composition according to any one of items 35 to 40,        wherein said peptide is diluted in HSA saline at a final        concentration of 0.2 mM, the resulting solution having a pH of        about 6.7.

    -   39. A method of inhibiting one or more of o) GIP-induced        glucagon secretion, i) GIP-induced insulin secretion, ii)        GIP-induced somatostatin secretion, iii) GIP-induced glucose        uptake, iv) GIP-induced fatty acid synthesis and/or fatty acid        incorporation, v) high or increased expression or activity of a        GIPR and vi) release of GIP following a meal (post-prandial GIP        release), said method comprising administering to an individual        in need thereof an effective amount of a peptide, a nucleic acid        construct, or a composition according to any one of the        preceding items.

    -   40. The peptide, the nucleic acid construct, or the composition        according to any one of the preceding items for use as a        medicament.

    -   41. The peptide, the nucleic acid construct, or the composition        according to any one of the preceding items for use in a method        of treating metabolic disorders.

    -   42. The peptide, the nucleic acid construct, or the composition        according to any one of the preceding items, wherein the        metabolic disorder is selected from the group consisting of        obesity, diabetes mellitus, insulin resistance, atherosclerosis,        and fatty acid metabolism disorder.

    -   43. The peptide, the nucleic acid construct, or the composition        according to any one of the preceding items for use in a method        of reducing serum levels of free fatty acids and/or serum levels        of triglycerides.

    -   44. The peptide, the nucleic acid construct, or the composition        according to any one of the preceding items for use in a method        of treating cancer, such as a cancer selected from the group        consisting of colon cancer, a neuroendocrine cancer and adrenal        adenoma.

    -   45. The peptide, the nucleic acid construct, or the composition        according to any one of the preceding items for use in a method        of treating a bone density disorder.

    -   46. The peptide, the nucleic acid construct, or the composition        according to any one of the preceding items, wherein the bone        density disorder is selected from the group consisting of        disorders characterized by low bone density and/or reduced bone        volume, disorders characterized by high bone density and/or        increased bone volume, and osteoporosis.

    -   47. The peptide, the nucleic acid construct, or the composition        for use according to any one of the preceding items, wherein        said peptide, nucleic acid construct or composition is to be        administered at least once daily, such as once daily.

    -   48. The peptide for use according to any of the preceding items,        wherein said peptide is to be administered at a dosage of at        least 500 pmol/kg/min, such as at least 1000 pmol/kg/min, such        as at least 1200 pmol/kg/min, such as at least 1500 pmol/kg/min,        such as at least 2000 pmol/kg/min, such as at least 2500        pmol/kg/min, such as at least 5000 pmol/kg/min.

    -   49. The peptide for use according to any of the preceding items,        wherein said peptide is to be administered at a dosage of 500 to        5000 pmol/kg/min, such as 500 to 1000 pmol/kg/min, such as 1000        to 1500 pmol/kg/min, such as 1500 to 2000 pmol/kg/min, such as        2000 to 2500 pmol/kg/min, such as 2500 to 3000 pmol/kg/min, such        as 3000 to 4000 pmol/kg/min, such as 4000 to 5000 pmol/kg/min.

    -   50. The peptide for use according to any of the preceding items,        wherein said peptide is to be administered at a daily dosage of        at least 30000 pmol/kg, such as at least 60000 pmol/kg, such as        at least 72000 pmol/kg, such as at least 90000 pmol/kg, such as        at least 120000 pmol/kg, such as at least 150000 pmol/kg.

    -   51. The peptide for use according to any of the preceding items,        wherein said peptide is to be administered by infusion.

    -   52. A kit of parts comprising a peptide, a nucleic acid        construct or a composition according to any of the preceding        items, and at least one additional component.

EXAMPLES Example 1—Materials and Methods

Materials

Human GIP was purchased from Bachem (H5645) Rat-GIP (027-12), while theremaining ligands were synthesized by Caslo™, Lyngby, Denmark. cDNA ofthe human GIP receptor was purchased from Origene (SC110906) and clonedinto the pCMV-Script vector. Iodinated human GIP was purchased fromPerkinElmer Life Sciences (NEX402025UC).

Animals

Male Wistar rats (220-250 g) were purchased from Charles RiverLaboratories more than 1 week before the experiments were performed, andgiven free access to standard rodent chow and water. Animals were housedtwo per cage and were subjected to a 12:12 h light-dark cycle.

Transfections and Tissue Culture

COS-7 cells were cultured at 10% C02 and 37° C. in Dulbecco's modifiedEagle's medium 1885 supplemented with 10% fetal bovine serum, 2 mMglutamine, 180 units/ml penicillin, and 45 g/ml streptomycin. Transienttransfection of the COS-7 cells for cAMP accumulation and competitionbinding was performed using the calcium phosphate precipitation methodwith the addition of chloroquin^(46, 47.)

cAMP Assay

In white 96-well plates transient transfected COS-7 cells were seededout in a density of 3*10⁴/well. The day after, the cells were washedtwice with Hepes buffered saline (HBS) buffer and incubated with HBS and1 mM 3-isobutyl-1-methylxanthine (IBMX) for 30 min at 37° C. To testagonists, ligands were added and incubated for 30 min at 37° C. In orderto test for antagonistic properties, the antagonist was preincubated for10 min and then the agonist was added and incubated for 20 additionalmin. The HitHunter™ cAMP XS assay (DiscoveRx) was carried out accordingto the manufacturer's instructions.

125I-Human GIP Competition Binding Assay

COS-7 cells were seeded in clear 96-well plates the day aftertransfection using a number of cells/well that obtained 5-10% specificbinding of the added radioactive ligand. The following day, cells wereassayed by competition binding for 4 h at 4° C. using 15-40 μM 125I-human GIP as well as unlabeled ligand in 50 mM Hepes buffer (pH 7.2)with 0.5% bovine serum albumin (BSA). After incubation, the cells werewashed twice in ice-cold binding buffer and lysed using 200 mM NaOH with1% SDS for 30 min. Nonspecific binding was determined as the binding oftracer to untransfected cells.

Isolated Perfused Rat or Mouse Pancreas

Non-fasted rats were anaesthetized with an IP injection ofHypnorm/Dormicum and the pancreas was dissected and perfused in situ.Briefly, the rat was killed by removal of the heart, and the pancreasperfused in a single-pass system through both the coeliac and thesuperior mesenteric artery via a catheter inserted into the adjacentabdominal aorta. All other aortic branches were ligated. The venouseffluent was collected for 1 min intervals via an obstructing cannulainserted into the portal vein, and stored at −20° C. until analysis. Theflow rate was kept constant at 4 ml/min. The perfusion medium wascontinuously gassed with a 95% O₂/5% CO₂ mixture to achieve pH 7.4, andmaintained at 37° C. during the entire experiment.

Hormone Analysis

Pancreatic somatostatin concentrations in venous effluent were analysedby RIA. Somatostatin immunoreactivity was determined using antiserum1758, which was raised in rabbits against synthetic cyclic somatostatinand recognizes both somatostatin-14 and somatostatin-28 [37, 38].

Example 2—GIP(1-30) is Found in T2DM Plasma Following a Meal

To verify if hGIP1-30 is indeed in human plasma, patients with T2DM weregiven a meal and hGIP1-30 was measured (FIG. 2). Compared to the mealinduced GIP1-42 response in T2DM patients⁴⁸ the hGIP(1-30) responsedisplays accelerated kinetics, however at a much lower concentration.Nevertheless, there is a clear hGIP1-30 response to a meal.

Example 3—Discovery of High Affinity Ligands of the hGIPR

In order to investigate the binding properties of variations of thehGIP3-30, heterologous competition binding experiments were conductedand compared to that of hGIP1-42. As seen in FIG. 3, the testedvariations of hGIP3-30 were able to displace ¹²⁵I-labeled hGIP with IC50values of 2 nM, 5.2 nM, 7.8 nM, 11.4 nM, and 16 nM (hGIP3-30-H18A,hGIP3-30-H18R, hGIP3-30-H18K, hGIP3-30-H18R+K30R, hGIP3-30,respectively). When compared with the IC50 value of 0.92 for homologuescompetition binding of hGIP, it is apparent that these are high affinityligands of the hGIPR.

Example 4—Variants of GIP(3-30) are High Affinity CompetitiveAntagonists

In order to evaluate potential antagonistic properties, hGIP1-42 inducedcAMP dose-response curves were made with increasing concentrations ofthe GIP3-30 variants and the corresponding Schild plot analysis wasmade. As seen in FIG. 4A/C/E, there are rightward shifts ofhGIP1-42-mediated cAMP response clearly displaying antagonisticcharacteristics. The linearity of the Schild plot analyses in FIG.4B/D/F, demonstrate a competitive nature of the GIP3-30 variants withcorresponding Ki values of 15 nM, 14 nM, and 54 nM for hGIP3-30,GIP3-30-H18R, and GIP3-30-H18R+K30R, respectively.

Example 5—hGIP(3-30) Antagonizes hGIP Induced Somatostatin Secretion inPerfused Rat Pancreata

Using perfused rat pancreata, it was demonstrated that hGIP3-30 wascapable of antagonizing somatostatin secretion induced by hGIP1-42 (FIG.5). hGIP1-42 alone induced a robust somatostatin secretion whilerGIP3-30 displayed a negligible response. Preincubation with hGIP3-30before the addition of hGIP1-42 led to a significantly reducedsomatostatin release, displaying in vivo efficacy.

Here we show that GIP1-30 is found under physiological conditionsfollowing a mixed meal in T2DM patients, underlining the potential ofthe naturally occurring antagonist, GIP3-30 in humans. Our bindingexperiments demonstrate that the tested variations of hGIP3-30 have ahigh affinity for the hGIPR. Of the functionally tested variations,hGIP3-30 and hGIP3-30-H18R are the most promising antagonist candidates.In perfused rat pancreata both antagonists displayed attenuation of GIPinduced somatostatin release.

Example 6—Formulation of hGIP3-30 (Acetate Salt) for Clinical Use

The peptide was ordered from Polypeptide laboratories and was formulatedas an acetate salt. For clinical use it is needed in solution with aphysiological pH. To achieve this 1 mM HCl, 0.1% human serum albumin(HSA) (3.4 pH) was added to the final concentration 1.9 mg/ml ofhGIP3-30. A further dilution with 0.2% HSA saline to 0.0162 mg/ml, wasdone to approximate the concentrations needed in the clinic. Thisresulted in a pH of 6.67, which is acceptable for clinical use. In orderto verify that no hGIP3-30 is lost following filtering and/orfreezing-cycles, hGIP3-30 concentrations were measured using a radioimmunoassay following various setups of filtering and/or freezing.Following solubility optimization, the hGIP3-30 is sent to the hospitalpharmacy formulation. hGIP3-30 in solution are aliquoted into 3 ml vialswith the final concentration of 0.2 mM. Finally, the patients receive1000 pmol/kg/min over an hour's duration.

Example 7—Characterization of hGIP Truncations

GIP(1-30)NH₂ is a naturally occurring truncation of GIP(1-42). Here wecharacterize eight N-terminal truncations of human GIP(1-30)NH₂: GIP(2-to 9-30)NH₂.

GIP(1-30)NH₂ is a naturally occurring truncation of GIP(1-42). Here wecharacterize eight N-terminal truncations of human GIP(1-30)NH₂: GIP(2-to 9-30)NH₂. Key results: GIP(1-30)NH₂ displaced ¹²⁵I-GIP(1-42) equallyto GIP(1-42) (Ki 0.75 nM), whereas the eight variants displayed loweraffinities (Ki 2.3-347 nM) with highest affinities of GIP(3-30)NH₂ and(5-30)NH₂. Agonism was only observed for GIP(1-30)NH₂ with an E_(max) on100% of GIP(1-42) and GIP(2-30)NH₂ (E_(max) 20%). GIP(2- to 9-30)NH₂displayed antagonism (IC₅₀ 12-450 nM) and right-shifts of theGIP(1-42)-response curve. Schild plot analyses identified GIP(3-30)NH₂and GIP(5-30)NH₂ as competitive antagonists (Ki 15 nM). Importantly,GIP(3-30) antagonized with a 26-fold higher potency than GIP(3-42).Binding studies with agonist (¹²⁵I-GIP(1-30)NH₂), partial agonist(¹²⁵I-GIP(2-30)NH₂) and competitive antagonist (¹²⁵I-GIP(3-30)NH₂)revealed distinct receptor conformations for these three ligand classes.The N-terminus is crucial for GIP functionality as agonist. Removal ofthe C-terminus of the naturally occurring DPP4-product GIP(3-42) createsanother naturally occurring, but superior antagonist GIP(3-30)NH₂, thattogether with GIP(5-30)NH₂ were high-affinity competitive antagonists.

Methods

Wild type human GIP receptor cDNA was purchased from Origene™,Rockville, Md., USA (SC110906) and cloned into the pCMV Script-vector.Human native GIP(1-42) was purchased from Bachem™, Bubendorf,Switzerland (H5645). All truncated GIP peptides were synthesized byCaslo™, Lyngby, Denmark and based on the human GIP sequence. PorcineGIP(3-42) was custom synthesized by PolyPeptide Laboratories(WolfenBüttel, Germany). ¹²⁵I-labelled native GIP (1-42) was purchasedfrom PerkinElmer Life Sciences, Skovlunde, Denmark (NEX402025UC). HumanGIP(1-30)NH₂, GIP(2-30)NH₂ and GIP(3-30)NH₂ were ¹²⁵I-labeled using thestandard stoichiometric chloramine T method as described previously(Holst and Bersani, 1991). The labeled peptides were purified byhigh-pressure liquid chromatography.

Cell Line and Transfection

COS-7 cells were grown in 10% CO₂ and at 37° C. in Dulbecco's modifiedEagle's medium 1885 supplemented with 10% fetal bovine serum, 2 mMglutamine, 180 units/ml penicillin, and 45 g/ml streptomycin.Transfection of COS-7 cells was performed using the calcium phosphateprecipitation method with chloroquine addition as previously described(Kissow et al., 2012).

cAMP-Assay

COS-7 cells (30.000 cells/well) were seeded in 96-well plates one daybefore transfection with human GIP receptor cDNA. Two days aftertransfection the cells were washed once with HEPES buffered saline (HBS)and incubated with HBS and 0.5 mM 3-isobutyl-1-methylxanthine (IBMX) for30 minutes at 37° C. The various truncated GIP variants were added tothe cells and incubated for 30 minutes at 37° C. in order to test forintrinsic activity. To test for antagonism of a given GIP variant, thecells were preincubated for 10 minutes at 37° C. with the GIP analoguefollowed by 20 minutes of incubation with GIP(1-42). The potency of theantagonists was determined from dose-response curves of the antagonistin the presence of a constant concentration of the GIP(1-42)corresponding to 50-80% of the maximal cAMP accumulation response(E_(max)) of GIP(1-42). For Schild analysis, various antagonistconcentrations were added 10 minutes prior to GIP(1-42) dose-responsecurves. After ligand incubation, the HitHunter™ cAMP XS assay (an enzymefragment complementation-based assay, DiscoveRx, Birmingham, UnitedKingdom) was carried out according to the manufacturer's instructions.All experiments were made in duplicates, and repeated at least threetimes. Luminescence was measured by Perkin Elmer™ EnVision 2104Multilabled reader (Skovlunde, Danmark). In brief, the cells were lysedin the wells, the enzyme fragment-cAMP-antibody, an enzyme fragment andthe enzyme substrates were added followed by 1 hour incubation at roomtemperature on shaker tray. The other enzyme fragment was added to thewells and incubated for 4 hours on shaker tray followed by measurementsof luminescence. The ligand-induced cAMP competed with the binding ofantibody to the first enzyme fragment and left the two fragments tofuse. The enzyme complex hydrolyzed the substrates and yieldedluminescence. The number “n” refers to individual experiments withseparate transfections although from same cell line.

Competitive Binding-Assay

COS-7 cells were seeded in 96-well plates 1 day after transfection withhuman GIP receptor cDNA. The number of cells seeded per well wasselected to result in 5-10% specific binding of the added radioactiveligand (1000-5000 cells/well). Two days after transfection, cells wereused for competition binding for 3 h at 4° C. to inhibit receptorinternalization using 6-10 μM/well of ¹²⁵I-GIP(1-42), ¹²⁵I-GIP(1-30)NH₂,¹²⁵I-GIP(2-30)NH₂, or ¹²⁵I-GIP(3-30)NH₂ as well as relevant amounts ofunlabeled ligands in 50 mM Hepes buffer, pH 7.4, supplemented with 0.5%(w/v) bovine serum albumin. After incubation for 3 hours at 4° C., thecells were washed twice in ice-cold binding buffer and lysed using 200mM NaOH with 1% SDS for 30 minutes. Nonspecific binding was determinedas the binding of radioligand to untransfected cells. All determinationswere made in duplicates, and all experiments repeated at least threetimes. The samples were analyzed for radioactivity using a Wallac Wizard1470 Gamma Counter (GMI Inc., Minnesota, USA). The number “n” refers toindividual experiments with separate transfections although from samecell line.

Data Analysis

IC₅₀, EC₅₀, and K_(d)/K_(i) values were determined by nonlinearregression. These, as well as maximal binding capacity (B_(max)) valuesand Schild plot analysis were carried out with the GraphPad Prism 6.0software (GraphPad, San Diego, Calif., USA) and Microsoft Excel™.Statistical analyses (unpaired t-tests) of two parameters were alsoperformed with GraphPad Prism 6.0. The calculations of B_(max) and K_(i)values were based on the formula for one class of binding sites inhomologous competition binding studies and the Cheng Prussoffs formula,respectively (DeBlasi et al., 1989). K_(d) is the dissociation constantdetermined by homologous receptor binding. Dose ratios (DR) for theSchild analyses were based on the potency shift of the GIP(1-42)dose-response curve in absence or presence of a fixed antagonistconcentration (DR=EC₅₀ in presence of antagonist/EC₅₀ in absence ofantagonist). Schild plots were performed with log (DR-1) (ordinate) andlog(antagonist concentration) (abscissa) to estimate the slopes and K,values (Lazareno and Birdsall, 1993).

Results

GIP(1-30)NH₂ is a full GIP receptor agonist with high affinity equal tonative GIP(1-42) To establish the role of the C-terminus for agonism inthe human GIP system, we first measured cAMP responses to humanGIP(1-42) and human GIP(1-30)NH₂ in transiently transfected COS-7 cellsexpressing the human GIP receptor (FIG. 6A). GIP(1-30)NH₂ was a fullagonist on the GIP receptor with a high potency (EC₅₀) of 11.2 μM [logEC₅₀-10.95±0.11], compared to the 6.0 μM [log EC₅₀-11.21±0.16] ofGIP(1-42) and with the same efficacy as GIP (1-42), consistent withearlier studies. Binding studies were performed with ¹²⁵I-GIP(1-42) asthe radioligand in the same cellular background. Truncation of the fulllength GIP(1-42) peptide at the 30-position did not change the affinityto the GIP receptor, and thus, resulted in affinities (IC₅₀) of 0.89 nMand 0.67 nM for GIP(1-30)NH₂ and GIP(1-42), respectively (FIG. 6B).Thus, GIP(1-30)NH₂ displayed the same potency, efficacy and affinity forthe human GIP receptor as GIP(1-42).

The N-Terminus is Essential for High Affinity Binding

To study the role of the N-terminus of human GIP(1-30)NH₂, the affinityof the eight N-terminally truncated peptides were compared to that ofGIP(1-30)NH₂ in transiently transfected COS-7 cells using ¹²⁵I-GIP(1-42)as radioligand (FIG. 7). Truncation resulted in decreased affinity witha tendency towards length-dependency, with a span from 2.3 fold to 347fold decrease in affinity compared to GIP(1-30)NH₂. GIP(3-30)NH₂followed by GIP(5-30)NH₂ displayed the highest affinities, whileGIP(9-30)NH₂ and GIP(6-30)NH₂ had more than 300 fold lower affinitiescompared to GIP(1-30)NH₂. Taken together, this emphasizes the importanceof the N-terminus for receptor binding.

GIP(2-30)NH₂ is a Partial Agonist and GIP(3- to 9-30)NH₂ are Antagonistsof the GIP Receptor

We measured cAMP accumulation in COS-7 cells, transiently transfectedwith the human GIP receptor, after incubation with each of the GIPvariants (FIGS. 8A and 8B). Removal of the first amino acid fromGIP(1-30)NH₂, created GIP(2-30)NH₂, which is a weak partial agonist withan efficacy of 20% compared to GIP(1-30)NH₂ and a potency of 3.7 nM [logEC₅₀-8.43±0.33, n=8] which is >3000 fold lower than GIP(1-30)NH₂.Removal of the second amino acid completely eliminated intrinsicactivity (FIG. 8A); a pattern that was also seen for the remainingtruncations (FIG. 8B). To determine whether the inactive forms hadantagonistic properties, increasing concentrations of the GIP variantswere added to a submaximal (50-80%) activation by GIP(1-42). All wereable to inhibit the cAMP response induced by GIP(1-42) (FIGS. 8C and8D). The most potent antagonists were GIP(3-30)NH₂ and GIP(5-30)NH₂ withIC50 of 11.8 nM and 11.9 nM respectively (Table 1) in agreement withtheir high binding affinities. Similar to the binding studies, theshortest GIP variant, GIP(9-30)NH₂, had the lowest antagonistic potencywith a 38-fold right-shift compared to GIP(3-30)NH₂.

TABLE 1 The table displays the IC₅₀-values from the binding studies(FIG. 7) with the fold change of GIP(1-30)NH₂ affinity, and the cAMPaccumulation studies (FIG. 8) with antagonist properties. Competitivebinding cAMP accumulation logIC ± SEM Ki (nM) fold n logIC₅₀ ± SEM IC₅₀(nM) n GIP(1-30)NH₂ −9.05 ± 0.02 0.89 1.0 13 — — — GIP(2-30)NH₂ −7.85 ±0.04 14.3 16 10 −7.66 ± 0.1 21.7 4 GIP(3-30)NH₂ −8.63 ± 0.04 2.3 2.6 12 −7.93 ± 0.04 11.8 6 GIP(4-30)NH₂ −7.67 ± 0.02 21.5 24 3 −6.97 ± 0.4 1084 GIP(5-30)NH₂ −8.23 ± 0.05 5.9 6.6 3 −7.92 ± 0.4 11.9 4 GIP(6-30)NH₂−6.46 ± 0.09 347 391 10 −6.47 ± 0.6 342 4 GIP(7-30)NH₂ −7.58 ± 0.08 2630 9 −6.86 ± 0.4 137 7 GIP(8-30)NH₂ −7.10 ± 0.04 79 89 3 −6.88 ± 0.5 1335 GIP(9-30)NH₂ −6.51 ± 0.08 307 345 3 −6.35 ± 0.6 450 4

GIP(3-30)NH₂ and GIP(5-30)NH₂ are Competitive Antagonists

A Schild analysis was performed for the four most potent antagonists, inaddition to the previously described antagonists GIP(6-30)NH₂ andGIP(7-30)NH₂. This analysis determines whether an antagonist actscompetitively and is illustrated by the Schild Plot. A straight linewith a Hill slope of 1.0 indicates competitive antagonism. Theantagonists were added in various constant concentrations to thedose-response curves of GIP(1-42) (FIG. 9). All six antagonists wereable to right-shift the GIP(1-42) dose-response curve with no changes inefficacy. However, only GIP(3-30)NH₂ and GIP(5-30)NH₂ act as purecompetitive antagonists judged by a straight line with a slope of 1(inserts in FIGS. 9B and 9D). These two ligands displayed slopes of0.93±0.02 and 1.1±0.04, respectively, while the slopes for GIP(2-30)NH₂,GIP(4-30)NH₂, GIP(6-30)NH₂, and GIP(7-30)NH₂ were 0.49±0.14, 0.75±0.02,0.38±0.13, and 0.17±0.03, respectively (FIG. 9B-F). The lack of abilityto compete equally with the agonist could indicate an allostericcomponent in the antagonistic properties of these ligands. TheX-intercept or pA₂-value of the Schild plot corresponds to the affinityconstant of the antagonist if the Hill slope equals 1. For the twocompetitive antagonists GIP(3-30)NH₂ and GIP(5-30)NH₂, the pA₂-valueswere 14.9 nM and 15.2 nM, respectively, thus in the same range as theK_(i) determined from the binding studies (2.3 nM and 5.9 nMrespectively). In summary, this analysis identified GIP(3-30)NH₂ andGIP(5-30)NH₂ as high affinity competitive GIP receptor antagonists.

The Functionalities of the Ligands Reflect the Binding Properties

The N-terminal truncations of GIP(1-30)NH₂ had a span in affinities (Ki)from 1 nM to 350 nM (FIG. 7 and Table 1) and concomitantly, displayeddifferent pharmacodynamics with both competitive and non-competitiveantagonistic properties (FIGS. 8 and 9). To further analyse the receptorinteraction of these variants we performed homologous competitivebinding studies with ¹²⁵I-GIP(1-30)NH₂, ¹²⁵I-GIP(2-30)NH₂, and¹²⁵I-GIP(3-30)NH₂ as radioligands (representing a full agonist, apartial agonist, and a competitive antagonist, respectively). The K_(d)values for GIP(1-30)NH₂, GIP(2-30)NH₂ and GIP(3-30)NH₂ obtained from thehomologous binding experiments (FIG. 10 and Table 2) were in the samerange as the Ki values obtained in the heterologous binding experimentsusing ¹²⁵I-GIP(1-42) as radioligand (Table 1). However, minor, yetsignificant, changes were observed upon a closer look at the affinities,as higher affinities were observed when GIP(1-30)NH₂ and GIP(2-30)NH₂competed with their own iodinated versions (homologous binding),compared to when they competed with ¹²⁵I-GIP(1-42) (heterologousbinding) (p=0.012 and p=0.0031, respectively, FIGS. 10A and B). Thus,the lack of C-terminus decreased the ability of GIP(1-30)NH₂ andGIP(2-30)NH₂ to compete with the full length agonist GIP(1-42) for theGIP receptor. In contrast, the N-terminally truncated antagonistGIP(3-30)NH₂, was able to displace the homologous radioligand with thesame affinity as the full agonist ¹²⁵I-GIP(1-42) radioligands (p=0.45,FIG. 10C). The B_(max) was calculated from the homologous bindingstudies (DeBlasi et al., 1989) and uncovered significantly more bindingsites for the antagonists compared to the two agonists (FIG. 10D), whichillustrates the general property of antagonists to stabilize severalinactive receptor confirmations, while agonists preferentially bind tothe active confirmation(s) (Rosenkilde et al., 1994).

TABLE 2 A ¹²⁵I-GIP(1-30)NH₂ log IC₅₀ fold change (IC₅₀) ±SEM (nM)GIP(1-30)NH₂ n GIP(1-42)NH₂ −9.24 0.19 0.58 1.9 3 GIP(1-30)NH₂ −9.520.16 0.30 1.0 5 GIP(2-30)NH₂ −7.59 0.18 26 84.3 4 GIP(3-30)NH₂ −8.350.071 4.4 14.5 4 GIP(6-30)NH₂ −5.97 0.066 1.065 3502 5 GIP(7-30)NH₂−7.43 0.25 37 120.9 5 B ¹²⁵I-GIP(2-30)NH₂ log IC₅₀ fold change (IC₅₀)±SEM (nM) GIP(1-30)NH₂ n GIP(1-42)NH₂ −9.36 0.087 0.43 0.9 3GIP(1-30)NH₂ −9.32 0.482 0.48 1.0 3 GIP(2-30)NH₂ −8.57 0.28 2.7 10.5 5GIP(3-30)NH₂ −9.12 0.20 0.76 1.6 3 GIP(6-30)NH₂ −6.47 0.28 340 707 4GIP(7-30)NH₂ −7.54 0.23 29 60.6 5 C ¹²⁵I-GIP(3-30)NH₂ log IC₅₀ foldchange (IC₅₀) ±SEM (nM) GIP(1-30)NH₂ n GIP(1-42)NH₂ −8.97 0.0015 1.070.6 3 GIP(1-30)NH₂ −8.78 0.063 1.7 1.0 3 GIP(2-30)NH₂ −8.11 0.065 7.74.6 4 GIP(3-30)NH₂ −8.47 0.12 3.4 2.0 5 GIP(6-30)NH₂ −6.43 0.26 370 2234 GIP(7-30)NH₂ −7.68 0.16 21 12.7 5

The binding properties were further elucidated through heterologousbinding studies with ¹²⁵I-GIP(1-30)NH₂, ¹²⁵I-GIP(2-30)NH₂¹²⁵I-GIP(3-30)NH₂ displaced by GIP(1-42), GIP(1-30)NH₂, GIP(2-30)NH₂,and GIP(3-30)NH₂, and the previously described GIP(6-30)NH₂, andGIP(7-30)NH₂ (Table 2). Again, the agonists GIP(1-30)NH₂ and GIP(1-42)displaced the agonist radioligand (¹²⁵I-GIP(1-30)NH₂) most efficiently,while their affinities decreased in competition with the radiolabeledantagonists. The opposite was observed for the antagonists thatdisplaced the partial agonist (¹²⁵I-GIP(2-30)NH₂) and the antagonist(¹²⁵I-GIP(3-30)NH₂) radioligand with highest affinities. Thereby, whenlooking at apparent affinities, the largest effects of increasedtruncation of GIP(1-30)NH₂ were observed with the agonist as radioligandwith >3000-fold decrease in affinity of GIP(7-30)NH₂ compared toGIP(1-30)NH₂ measured with agonist radioligand, and only 223-folddecrease when measured with ¹²⁵I-GIP(3-30)NH₂ as radioligand. Thispattern was observed for all four antagonists (Table 2).

The C-Terminal Part of GIP Acts as a Negative Regulator of theAntagonistic Action of GIP(3-42)

The identification of GIP(3-30)NH₂ as the most potent antagonistprompted us to compare it with GIP(3-42) in order to directly determinethe impact of the C-terminal amino acids 31 through 42. We also includedthe porcine GIP(3-42), representing a low-potent antagonist on the humanGIP receptor in vitro, with no ability to antagonize porcineGIP(1-42)-mediated insulin secretion in pigs at physiologicalconcentrations (Deacon et al., 2006). Porcine GIP(3-42) has an argininein position 18 and serine in position 34, whereas the human sequence hashistidine and asparagine, respectively. Like GIP(3-30)NH₂ (FIG. 8A),neither of the GIP(3-42) variants had any intrinsic agonistic activityin cAMP-accumulation assay (data not shown, n=3), but both were able toantagonize submaximal (50-80%) human GIP(1-42)-induced activation (FIG.11B). Importantly, human GIP(3-42) was remarkably less potent than humanGIP(3-30)NH₂ (26-fold lower potency, FIG. 11B) and 1 μM of this resultedin only 7.3-fold shift in the dose-response curve of human GIP(1-42)compared to 281 fold for human GIP(3-30)NH₂ (FIG. 11C). The porcinevariant displayed higher potency compared to human GIP (3-42), yet notas high as human GIP(3-30)NH₂. Thus, the C-terminus has a functionalrole as its absences improve the antagonistic properties in GIP(3-30)NH₂compared to GIP(3-42).

Example 8—Characterization of GIP Mutations/Variants

cAMP accumulation assays and schild plots were performed essentially asoutlined in the above examples. Selected mutations in the GIP3-30peptide were tested. Mutations of the amino acids in position 15 and 18(GIP(3-30) 15E18A) decreased the Ki-value and therefore increased theantagonistic capabilities. See FIGS. 13-15.

Hill- Experi- Ki slope of ments Name Mutations (nM) plot (number)hGIP(3-30) — 15 1.1 4 (SEQ ID NO: 1) 18R (Rat) Position 18 to Arginine14 0.6 4 (SEQ ID NO: 2) 18R30R (Mouse) Pos. 18 and 30 → 54 0.7 4 (SEQ IDNO: 3) Arginine 18A Position 18 to Alanine 35 0.9 3 (SEQ ID NO: 79) 18KPosition 18 to Lysine 37 0.6 4 (SEQ ID NO: 80) 15E18A Pos. 15 → Glutamic7.9 0.7 3 (SEQ ID NO: 81) acid and pos. 18 → Alanine 16A18A Pos. 16 and18 → 15 0.5 3 (SEQ ID NO: 82) Alanine

Example 9

Studies in rodents are conducted to verify the effect of hGIP(3-30) (SEQID NO:1) in vivo. The GIP receptor antagonist is administered in rats(n=8-10) before an oral glucose tolerance test (OGTT). The rats aregiven a glucose load after subcutaneous administration of the antagonistand the glucose tolerance will be measured by plasma concentrations ofglucose and insulin the following hour. The same procedure is previouslyperformed with other GIP antagonists (Pathak et al., 2015).

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Sequences Description Sequence 1 Human GIP3-30EGTFISDYSIAMDKIHQQDFVNWLLAQK (hGIP3-30) 2 Rat GIP3-30EGTFISDYSIAMDKIRQQDFVNWLLAQK (rGIP3-30); GIP(3-30)H18R; 3 Mouse GIP3-30EGTFISDYSIAMDKIRQQDFVNWLLAQR (mGIP3-30): GIP(3- 30)H18R/K30R 4 GIP1-42YAEGTFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂GKKNDWK (consensus) HNITQ 5 GIP5-25TFISDYSIAMDKIX ₁QQDFVNW 6 GIP3-25 EGTFISDYSIAMDKIX ₁QQDFVNW 7 GIP3-26EGTFISDYSIAMDKIX ₁QQDFVNWL 8 GIP3-27 EGTFISDYSIAMDKIX ₁QQDFVNWLL 9GIP3-28 EGTFISDYSIAMDKIX ₁QQDFVNWLLA 10 GIP3-29 EGTFISDYSIAMDKIX₁QQDFVNWLLAQ 11 GIP3-30 EGTFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂ (consensus) 12GIP3-31 EGTFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂G 13 GIP3-32 EGTFISDYSIAMDKIX₁QQDFVNWLLAQX ₂GK 14 GIP3-33 EGTFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂GKK 15GIP3-34 EGTFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂GKKN 16 GIP3-35 EGTFISDYSIAMDKIX₁QQDFVNWLLAQX ₂GKKND 17 GIP3-36 EGTFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂GKKNDW18 GIP3-37 EGTFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂GKKNDWK 19 GIP3-38EGTFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂GKKNDWKH 20 GIP3-39 EGTFISDYSIAMDKIX₁QQDFVNWLLAQX ₂GKKNDWKHN 21 GIP3-40 EGTFISDYSIAMDKIX ₁QQDFVNWLLAQX₂GKKNDWKHNI 22 GIP3-41 EGTFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂GKKNDWKHNIT 23GIP4-25 GTFISDYSIAMDKIX ₁QQDFVNW 24 GIP4-26 GTFISDYSIAMDKIX ₁QQDFVNWL 25GIP4-27 GTFISDYSIAMDKIX ₁QQDFVNWLL 26 GIP4-28 GTFISDYSIAMDKIX₁QQDFVNWLLA 27 GIP4-29 GTFISDYSIAMDKIX ₁QQDFVNWLLAQ 28 GIP4-30GTFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂ (consensus) 29 GIP4-31 GTFISDYSIAMDKIX₁QQDFVNWLLAQX ₂G 30 GIP4-32 GTFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂GK 31 GIP4-33GTFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂GKK 32 GIP4-34 GTFISDYSIAMDKIX₁QQDFVNWLLAQX ₂GKKN 33 GIP4-35 GTFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂GKKND 34GIP4-36 GTFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂GKKNDW 35 GIP4-37 GTFISDYSIAMDKIX₁QQDFVNWLLAQX ₂GKKNDWK 36 GIP4-38 GTFISDYSIAMDKIX ₁QQDFVNWLLAQX₂GKKNDWKH 37 GIP4-39 GTFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂GKKNDWKHN 38 GIP4-40GTFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂GKKNDWKHNI 39 GIP4-41 GTFISDYSIAMDKIX₁QQDFVNWLLAQX ₂GKKNDWKHNIT 40 GIP4-42 GTFISDYSIAMDKIX ₁QQDFVNWLLAQX₂GKKNDWKHNITQ 41 GIP5-26 TFISDYSIAMDKIX ₁QQDFVNWL 42 GIP5-27TFISDYSIAMDKIX ₁QQDFVNWLL 43 GIP5-28 TFISDYSIAMDKIX ₁QQDFVNWLLA 44GIP5-29 TFISDYSIAMDKIX ₁QQDFVNWLLAQ 45 GIP5-30 TFISDYSIAMDKIX₁QQDFVNWLLAQX ₂ (consensus) 46 GIP5-31 TFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂G47 GIP5-32 TFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂GK 48 GIP5-33 TFISDYSIAMDKIX₁QQDFVNWLLAQX ₂GKK 49 GIP5-34 TFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂GKKN 50GIP5-35 TFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂GKKND 51 GIP5-36 TFISDYSIAMDKIX₁QQDFVNWLLAQX ₂GKKNDW 52 GIP5-37 TFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂GKKNDWK53 GIP5-38 TFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂GKKNDWKH 54 GIP5-39TFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂GKKNDWKHN 55 GIP5-40 TFISDYSIAMDKIX₁QQDFVNWLLAQX ₂GKKNDWKHNI 56 GIP5-41 TFISDYSIAMDKIX ₁QQDFVNWLLAQX₂GKKNDWKHNIT 57 GIP5-42 TFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂GKKNDWKHNITQ 58Consensus EGTFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂GKKNDWKHNI GIP3-42 TQ 59hGIP5-25 TFISDYSIAMDKIHQQDFVNW 60 mGIP5-25 and TFISDYSIAMDKIRQQDFVNWrGIP5-25 61 GIP5-25 H18A TFISDYSIAMDKIAQQDFVNW 62 hGIP3-42EGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ 63 rGIP3-42EGTFISDYSIAMDKIRQQDFVNWLLAQKGKKNDWKHNITQ 64 mGIP3-42EGTFISDYSIAMDKIRQQDFVNWLLAQRGKKNDWKHNIT 0 65 hGIP1-42YAEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKH NITQ 66 rGIP1-42YAEGTFISDYSIAMDKIRQQDFVNWLLAQKGKKNDWKH NITQ 67 mGIP1-42YAEGTFISDYSIAMDKIRQQDFVNWLLAQRGKKNDWKH NITQ 68 GIP1-30YAEGTFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂ (consensus) 69 hGIP1-30YAEGTFISDYSIAMDKIHQQDFVNWLLAQK 70 rGIP1-30YAEGTFISDYSIAMDKIRQQDFVNWLLAQK 71 mGIP1-30YAEGTFISDYSIAMDKIRQQDFVNWLLAQR 72 GIP5-25 (H to K) TFISDYSIAMDKIKQQDFVNW73 GIP5-25 (variant) TFISDYSIAMX _(0a) X 0bIX ₁QQDFVNW 74 GIP3-30EGTFISDYSIAMX _(0a) X 0bIX ₁QQDFVNWLLAQX ₂ (variant) 75 GIP4-30GTFISDYSIAMX _(0a) X 0bIX ₁QQDFVNWLLAQX ₂ (variant) 76 GIP5-30TFISDYSIAMX0,X0b1X1QQDFVNWLLAQX ₂ (variant) 77 hGIP4-30GTFISDYSIAMDKIHQQDFVNWLLAQK 78 hGIP5-30 TFISDYSIAMDKIHQQDFVNWLLAQK 79hGIP(3-30)H18A EGTFISDYSIAMDKIAQQDFVNWLLAQK 80 hGIP(3-30)H18KEGTFISDYSIAMDKIKQQDFVNWLLAQK 81 hGIP(3-30) EGTFISDYSIAMEKIAQQDFVNWLLAQKD15EH18A 82 hGIP(3-30) EGTFISDYSIAMDAIAQQDFVNWLLAQK K16AH18A 83hGIP(3-30)D15E EGTFISDYSIAMEKIHQQDFVNWLLAQK 84 hGIP(3-30)D15NEGTFISDYSIAMNKIHQQDFVNWLLAQK 85 hGIP(3-30)K16AEGTFISDYSIAMDAIHQQDFVNWLLAQK 86 hGIP(3-30)K16HEGTFISDYSIAMDHIHQQDFVNWLLAQK 87 hGIP(3-30)K16REGTFISDYSIAMDRIHQQDFVNWLLAQK 88 hGIP(3-30)H18FEGTFISDYSIAMDKIFQQDFVNWLLAQK 89 hGIP(3-30)H18WEGTFISDYSIAMDKIWQQDFVNWLLAQK 90 hGIP(3-30)K30REGTFISDYSIAMDKIHQQDFVNWLLAQR 91 hGIP(3-30)K30HEGTFISDYSIAMDKIHQQDFVNWLLAQH

1.-28. (canceled)
 29. A peptide selected from the group consisting of: apeptide consisting of 28 contiguous amino acids of sequence(GIP3-30; SEQ ID NO: 74) EGTFISDYSIAMX _(0a) X _(0b)IX ₁QQDFVNWLLAQX ₂,

a peptide consisting of 27 contiguous amino acids of sequence(GIP4-30; SEQ ID NO: 75) GTFISDYSIAMX _(0a) X _(0b)IX ₁QQDFVNAVLLAQX ₂,

a peptide consisting of 26 contiguous amino acids of sequence(GIP5-30; SEQ ID NO: 76) TFISDYSIAMX _(0a) X _(0b)IX ₁QQDFVNAVLLAQX ₂,

wherein X_(0a), X_(0b), X₁ and X₂ are individually any amino acid, and avariant of any of the above peptides having at least 80% sequenceidentity to said peptide, wherein said peptide or variant thereof is anantagonist of a GIP receptor (GIPR), with the proviso that said peptideor variant thereof does not have 100% sequence identity to nativehGIP3-30 (SEQ ID NO: 1), to native hGIP4-30 (SEQ ID NO: 77) or to nativehGIP5-30 (SEQ ID NO: 78).
 30. The peptide according to claim 29, whereinX_(0a) is selected from the group consisting of D and E; X_(0b) isselected from the group consisting of K, A, H, and R; X₁ is selectedfrom the group consisting of H, R, A, K, F, and Y; and X₂ is selectedfrom the group consisting of K, R, H, and A.
 31. The peptide accordingto claim 29, wherein the amino acid sequence of said peptide or variantthereof differs from SEQ ID NO: 74, SEQ ID NO: 75 or SEQ ID NO: 76, inthat the amino acid sequence of the variant comprises one, two, three,four, or five amino acid substitutions.
 32. The peptide according toclaim 29, wherein the amino acid sequence of said peptide or variantthereof differs from SEQ ID NO: 74, SEQ ID NO: 75 or SEQ ID NO: 76, inthat the amino acid sequence of the variant comprises one, two, three,or four amino acid substitutions.
 33. The peptide according to claim 29,wherein the amino acid sequence of said peptide or variant thereofdiffers from SEQ ID NO: 74, SEQ ID NO: 75 or SEQ ID NO: 76, in that theamino acid sequence of the variant comprises one, two, or three aminoacid substitutions.
 34. The peptide according to claim 29, wherein theamino acid sequence of said peptide or variant thereof differs from SEQID NO: 74, SEQ ID NO: 75 or SEQ ID NO: 76, in that the amino acidsequence of the variant comprises one or two amino acid substitutions.35. The peptide according to claim 31, wherein said amino acidsubstitutions are selected from the group consisting of: i) substitutionof an amino acid having a polar side chain for a different amino acidhaving a polar side chain wherein amino acids having a polar side chainare Asp, Glu, Lys, Arg, His, Asn, Gln, Ser, Thr, Tyr, and Cys; ii)substitution of an amino acid having a non-polar side chain for adifferent amino acid having a non-polar side chain wherein amino acidshaving a non-polar side chain are Gly, Ala, Val, Leu, Be, Phe, Trp, Pro,and Met; iii) substitution of an amino acid having an aliphatic sidechain for a different amino acid having an aliphatic side chain whereinamino acids having a polar side chain are Gly, Ala Val, Leu, and Ile;iv) substitution of an amino acid having a cyclic side chain for adifferent amino acid having a cyclic side chain wherein amino acidshaving a cyclic side chain are Phe, Tyr, Trp, His, and Pro; v)substitution of an amino acid having an aromatic side chain for adifferent amino acid having an aromatic side chain wherein amino acidshaving an aromatic side chain are Phe, Tyr, and Trp; vi) substitution ofan amino acid having an acidic side chain for a different amino acidhaving an acidic side chain wherein amino acids having an acidic sidechain are Asp, and Glu; vii) substitution of an amino acid having abasic side chain for a different amino acid having a basic side chainwherein amino acids having a basic side chain are Lys, Arg, and His;viii) substitution of an amino acid having an amide side chain for adifferent amino acid having an amide side chain wherein amino acidshaving an amide side chain are Asn, and Gln; ix) substitution of anamino acid having a hydroxy side chain for a different amino acid havinga hydroxy side chain wherein amino acids having a hydroxy side chain areSer, and Thr; x) substitution of an amino acid having asulphur-containing side chain for a different amino acid having asulphur-containing side chain wherein amino acids having asulphur-containing side chain are Cys and Met; xi) substitution of aneutral, weakly hydrophobic amino acid for a different neutral, weaklyhydrophobic amino acid wherein neutral, weakly hydrophobic amino acidsare Pro, Ala, Gly, Ser, and Thr; xii) substitution of a hydrophilic,acidic amino acid for a different hydrophilic, acidic amino acid whereinhydrophilic, acidic amino acids are Gln, Asn, Glu, and Asp; and xiii)substitution of a hydrophobic amino acid for a different hydrophobicamino acid wherein hydrophobic amino acids are Leu, Ile, and Val. 36.The peptide according to claim 29, wherein said peptide is selected fromthe group consisting of: a peptide consisting of 28 contiguous aminoacids of sequence (GIP3-30; SEQ ID NO: 11) EGTFISDYSIAMDKIX₁QQDFVNAVLLAQX ₂,

a peptide consisting of 27 contiguous amino acids of sequence(GIP4-30; SEQ ID NO: 28) GTFISDYSIAMDKIX ₁QQDFVNAVLLAQX ₂,

a peptide consisting of 26 contiguous amino acids of sequence(GIP5-30; SEQ ID NO: 45) TFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂,

wherein X₁ is selected from the group consisting of H, R, A, K, F, andY; and X₂ is selected from the group consisting of K, R, H, and A; and avariant of any of the above peptides having at least 80% sequenceidentity to said peptide, with the proviso that said peptide or variantthereof does not have 100% sequence identity to native hGIP3-30 (SEQ IDNO: 1), to native hGIP4-30 (SEQ ID NO: 77) or to native hGIP5-30 (SEQ IDNO: 78); wherein said peptide or variant thereof is an antagonist of aGIP receptor (GIPR).
 37. The peptide according to claim 36, wherein theamino acid sequence of the variant differs from SEQ ID NO: 11, SEQ IDNO: 28 or SEQ ID NO: 45, in that the amino acid sequence of the variantcomprises one or two amino acid substitutions.
 38. The peptide accordingto claim 29, wherein said peptide is selected from the group consistingof: a peptide consisting of 28 contiguous amino acids of sequence(GIP3-30; SEQ ID NO: 74) EGTFISDYSIAMX _(0a) X _(0b)IX ₁QQDFVNWLLAQX ₂,or (GIP3-30; SEQ ID NO: 11) EGTFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂,

or a variant of any one of the above peptides, wherein the amino acidsequence of the variant differs from SEQ ID NO: 74 and SEQ ID NO: 11 inthat the amino acid sequence of the variant comprises one, two, three,four or five amino acid substitutions, wherein said peptide or variantthereof is an antagonist of a GIP receptor (GIPR), with the proviso thatsaid peptide or variant thereof does not have 100% sequence identity tonative hGIP3-30 (SEQ ID NO: 1).
 39. The peptide according to claim 38,wherein the amino acid sequence of the variant differs from SEQ ID NO:11, SEQ ID NO: 28 or SEQ ID NO: 45, in that the amino acid sequence ofthe variant comprises one or two amino acid substitutions.
 40. Thepeptide according to claim 29, wherein said peptide is selected from thegroup consisting of: (hGIP(3-30)H18A; SEQ ID NO: 79)EGTFISDYSIAMDKIAQQDFVNWLLAQK, (hGIP(3-30)H18K; SEQ ID NO: 80)EGTFISDYSIAMDKIKQQDFVNWLLAQK, (hGIP(3-30)D15EH18A; SEQ ID NO: 81)EGTFISDYSIAMEKIAQQDFVNWLLAQK, (hGIP(3-30)K16AH18A; SEQ ID NO: 82)EGTFISDYSIAMDAIAQQDFVNWLLAQK, (hGIP(3-30)D15E; SEQ ID NO: 83)EGTFISDYSIAMEKIHQQDFVNWLLAQK, (hGIP(3-30)D15N; SEQ ID NO: 84)EGTFISDYSIAMNKIHQQDFVNWLLAQK, (hGIP(3-30)K16A; SEQ ID NO: 85)EGTFISDYSIAMDAIHQQDFVNWLLAQK, (hGIP(3-30)K16H; SEQ ID NO: 86)EGTFISDYSIAMDHIHQQDFVNWLLAQK, (hGIP(3-30)K16R; SEQ ID NO: 87)EGTFISDYSIAMDRIHQQDFVNWLLAQK, (hGIP(3-30)H18F; SEQ ID NO: 88)EGTFISDYSIAMDKIFQQDFVNWLLAQK, (hGIP(3-30)H18W; SEQ ID NO: 89)EGTFISDYSIAMDKIWQQDFVNWLLAQK, (hGIP(3-30)K30R SEQ ID NO: 90)EGTFISDYSIAMDKIHQQDFVNWLLAQR, (hGIP(3-30)K30H; SEQ ID NO: 91)EGTFISDYSIAMDKIHQQDFVNWLLAQH,

and a variant of any one of the above peptides, wherein the amino acidsequence of the variant differs from SEQ ID NO: 79 to SEQ ID NO: 91 inthat the amino acid sequence of the variant comprises one, two, three,four or five amino acid substitutions.
 41. The peptide according toclaim 29, wherein said peptide is C-terminally amidated (—NH₂) and/orN-terminally acetylated (COCH₃).
 42. A nucleic acid construct encoding apeptide selected from the group consisting of a peptide consisting of 28contiguous amino acids of amino acid sequence (GIP3-30; SEQ ID NO: 74)EGTFISDYSIAMX _(0a) X _(0b)IX ₁QQDFVNWLLAQX ₂,

a peptide consisting of 27 contiguous amino acids of amino acid sequence(GIP4-30; SEQ ID NO: 75) GTFISDYSIAMX _(0a) X _(0b)IX ₁QQDFVNWLLAQX ₂,

a peptide consisting of 26 contiguous amino acids of amino acid sequence(GIP5-30; SEQ ID NO: 76) TFISDYSIAMX _(0a) X _(0b)IX ₁QQDFVNWLLAQX ₂,

wherein X_(0a), X_(0b), X₁ and X₂ are individually any amino acid, and avariant of any of the above peptides having at least 80% sequenceidentity to said peptide, wherein said peptide or variant thereof is anantagonist of a GIP receptor (GIPR).
 43. The nucleic acid constructaccording to claim 42, wherein X_(0a) is selected from the groupconsisting of D and E; X_(0b) is selected from the group consisting ofK, A, H, and R; X₁ is selected from the group consisting of H, R, A, K,F, and Y; and X₂ is selected from the group consisting of K, R, H, andA.
 44. The nucleic acid construct according to claim 42, wherein saidpeptide is selected from the group consisting of: a peptide consistingof 28 contiguous amino acids of sequence (GIP3-30; SEQ ID NO: 11)EGTFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂,

a peptide consisting of 27 contiguous amino acids of sequence(GIP4-30; SEQ ID NO: 28) GTFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂,

a peptide consisting of 26 contiguous amino acids of sequence(GIP5-30; SEQ ID NO: 45) TFISDYSIAMDKIX ₁QQDFVNWLLAQX ₂,

wherein X₁ is selected from the group consisting of H, R, A, K, F, andY; and X₂ is selected from the group consisting of K, R, H, and A; and avariant of any one of the above peptides, wherein the amino acidsequence of the variant differs from SEQ ID NO: 11, SEQ ID NO: 28 or SEQID NO: 45, in that the amino acid sequence of the variant comprises one,two, three, four or five amino acid substitutions, wherein said peptideor variant thereof is an antagonist of a GIP receptor (GIPR).
 45. Thenucleic acid construct according to claim 42, wherein said peptide isselected from the group consisting of: (hGIP3-30, SEQ ID NO: 1)EGTFISDYSIAMDKIHQQDFVNWLLAQK, (rGIP3-30, SEQ ID NO: 2)EGTFISDYSIAMDKIRQQDFVNWLLAQK, (mGIP3-30, SEQ ID NO: 3)EGTFISDYSIAMDKIRQQDFVNWLLAQR, (hGIP4-30, SEQ ID NO: 77)GTFISDYSIAMDKIHQQDFVNWLLAQK, (hGIP5-30, SEQ ID NO: 78)TFISDYSIAMDKIHQQDFVNWLLAQK,

and a variant of any one of the above peptides, wherein the amino acidsequence of the variant differs from SEQ ID NO: 1, SEQ ID NO: 2, SEQ IDNO: 3, SEQ ID NO: 77, or SEQ ID NO: 78, in that the amino acid sequenceof the variant comprises one, two, three, four or five amino acidsubstitutions, wherein said peptide or variant thereof is an antagonistof a GIP receptor (GIPR).
 46. The nucleic acid construct according toclaim 42, wherein said peptide is selected from the group consisting of(hGIP(3-30)H18A; SEQ ID NO: 79) EGTFISDYSIAMDKIAQQDFVNWLLAQK,(hGIP(3-30)H18K; SEQ ID NO: 80) EGTFISDYSIAMDKIKQQDFVNWLLAQK,(hGIP(3-30)D15EH18A; SEQ ID NO: 81) EGTFISDYSIAMEKIAQQDFVNWLLAQK,(hGIP(3-30)K16AH18A; SEQ ID NO: 82) EGTFISDYSIAMDAIAQQDFVNWLLAQK,(hGIP(3-30)D15E; SEQ ID NO: 83) EGTFISDYSIAMEKIHQQDFVNWLLAQK,(hGIP(3-30)D15N; SEQ ID NO: 84) EGTFISDYSIAMNKIHQQDFVNWLLAQK,(hGIP(3-30)K16A; SEQ ID NO: 85) EGTFISDYSIAMDAIHQQDFVNWLLAQK,(hGIP(3-30)K16H; SEQ ID NO: 86) EGTFISDYSIAMDHIHQQDFVNWLLAQK,(hGIP(3-30)K16R; SEQ ID NO: 87) EGTFISDYSIAMDRIHQQDFVNWLLAQK,(hGIP(3-30)H18F; SEQ ID NO: 88) EGTFISDYSIAMDKIFQQDFVNWLLAQK,(hGIP(3-30)H18W; SEQ ID NO: 89) EGTFISDYSIAMDKIWQQDFVNWLLAQK,(hGIP(3-30)K30R SEQ ID NO: 90) EGTFISDYSIAMDKIHQQDFVNWLLAQR, and(hGIP(3-30)K30H; SEQ ID NO: 91) EGTFISDYSIAMDKIHQQDFVNWLLAQH,

and a variant of any one of the above peptides, wherein the amino acidsequence of the variant differs from SEQ ID NO: 79 to SEQ ID NO: 91 inthat the amino acid sequence of the variant comprises one, two, three,four or five amino acid substitutions.
 47. The nucleic acid constructaccording to claim 42, wherein said nucleic acid construct is comprisedin a delivery vehicle.